APPARATUS AND METHOD FOR DETECTING AND CLASSIFYING CHEMICALS, PARTICLES, VIRA, AND BACTERIA IN FLUIDS

Abstract: The present invention is drawn to the detection of harmful agents, known or previously unknown, using a biosensor. The biosensor makes use of bioreceptors for detecting and classifying chemicals, poisons, particles, vira, and bacteria in fluids. In a preferred embodiment, the bioreceptor is either an aptamer or a poikilothermic and quiescent cell packaged in a collection device and treated to evoke an optical response to the presence of harmful agents, allowing for photocelss detection and signal processing. Harmful agents can also be collected in this manner for further analysis, and antibodies produced by the apparatus.

Description: TITLE: Apparatus and Method for Detecting and Classifying Chemicals, Particles, Vira, and Bacteria in Fluids FIELD OF THE INVENTION The present invention is drawn to the detection of harmful agents, or agents in general, using a biosensor. More particularly, it is drawn to the use of bioreceptors for detecting and classifying chemicals, poisons, particles, vira, and bacteria in fluids. In a preferred embodiment, a bioreceptor selected from the group consisting of aptameric compounds and poikilothermic and quiescent cells is packaged in a collection device and treated to evoke an optical response to the presence of such agents, allowing for photocell detection and subsequent optional signal processing. These agents can also be collected in this manner for further analysis. BACKGROUND INFORMATION Chemical and biological weapons, sometimes referred to as the "poor man's nuclear weapons," pose a significant threat in the post-Cold War environment. The relative low cost and simplicity of their design and technology, in comparison to nuclear weapons, make them the weapons of mass destruction choice for a variety of rogue states and terrorist, non-state organizations. This threat has been made all the more tangible by the use of chemical agent in a Tokyo subway, and allegations over Iraq's development of chemical and biological weapons and its actual use of chemical weapons in combat operations. According to the 1998 U.S Army Science and Technology Master Plan, "New fluorescent, acoustic, and optical biosensors are being designed for enhanced sensitivity and more flexible detection capability. Recent advances in the acceleration of the polymerase chain reaction (PCR) on a miniaturized scale now permit the exploitation of DNA probes for field detection of pathogens. A major thrust of a Joint Warfighting Science and Technology Plan (JWSTP) Defense Technology Obj ective (DTO), J.04 "Integrated Detection Advanced Technology Demonstration (ATD)," is the development of a rapid, automated field detection device based on the PCR. One key DTO element is the development of recombinant antibodies to serve as the recognition element of these new biosensors (FY98). Recombinant antibodies will ultimately be designed and quickly selected from genetic "super libraries" (FY99) to have specific detection capabilities, and novel starburst dendrimers are being studied for use on tailored reactive surfaces. Another major approach to point detection is mass spectrometry (MS), and miniature automated pyrolysis-based versions are being assessed for integration into existing CBD platforms (FY01). Of critical importance for biosensor and MS approaches is bio-aerosol sampling, since characteristics (e.g., concentration of detectable units per unit volume of air) of biological aerosols differ dramatically from chemical vapors, with resulting effects on detection efficacy." Of particular concern, which this invention addresses, are needs for (1) detecting, classifying and capturing agents heretofore undiscovered or unanalyzed, (2) annunciating these findings with a typical total processing time from exposure of about one second, (3) compactness and relatively low cost combined with long unattended life in the field, and (4) sensitivity to agents whose lethal or otherwise effective concentration in the environment will be extremely low compared to the comparable prior art. BRIEF SUMMARY OF THE INVENTION It is an object of the invention to use specialized cells for detecting and classifying chemicals, particles, vira, and bacteria in fluids. It is yet another object of the invention to provide a system for directing harmful agents onto a sensor cell surface for use in detecting and classifying chemicals, particles, vira, and bacteria in fluids. It is yet another object of the invention to provide diagnostic information and annunciation, either locally or remotely, within approximately 1 second or less of exposure to an agent in the free fluid, the time being short enough to become a function of the biochemical sensing apparatus alone. It is another object of the invention to provide a aptameric compound packaged in a collection device and treated to evoke an optical response to the presence of harmful agents, allowing for photocell detection and/or collection of the harmful agents. It is another object of the invention to provide a stationary poikilotherrnic cell with minimal environmental and metabolic requirements packaged in a collection device and treated to evoke an optical response to the presence of harmful agents, allowing for photocell detection and/or collection of the harmful agents. It is an object of the invention to provide a biosensor having a structure for sorting and selecting components of a fluid for acquisition and impingement on sensor cell surfaces for alarm and/or analysis purposes. It is an object of the invention to use a bioreceptor selected from the group consisting of aptamers, activatable heterotrophes, encysting ameba, epidermal cells, melanocytes, macrophages, phagic yeasts, and certain species of fungi for the detection of harmful agents. It is another object of the invention to provide the functions of acquisition, filtering, sorting, selection, impingement on sensor cell surfaces and trapping of particles in a fluid, in accordance with desired mathematical functions of size, density, shape and surface characteristics. It is another object of the invention to provide a combination of mechanical, aerodynamic, and kinetic methods to concentrate and/or sort particles of desired size and/or density from a fluid (gaseous, vapor or liquid) for detection by a sensor cell. It is another object of the invention to provide a combination of mechanical, aerodynamic, and kinetic methods to impinge particles of desired size and/or density onto a bioreceptor surface for detection. It is another object of the invention to provide a system for providing rapid annunciation of chemical, biological, and nuclear threats through interconnection of a plurality of biosensors via a central readout and decision system. It is yet another object of the invention to provide a biosensor using aptameric compounds. It is yet another object of the invention to provide a biosensor using a poikilothermic and quiescent cell selected from the group consisting of activatable heterotrophes, encysting ameba, epidermal cells, melanocytes, macrophages, phagic yeasts, and certain species of fungi. It is another object of this invention to provide broad annunciation capability combined with specificity, against agents that may not yet have been discovered by the emplacer of the biosensor It is another object of this invention to achieve a sensing and processing time of about one second from exposure. It is another object of this invention to combine compactness and low cost with long unattended life in the field. It is another object of this invention to provide sensitivity to agents at very low concentrations in the environment. It is a further object of this invention to provide for capturing the offending agents and furnishing a means for rapid preparation of antibodies to these agents. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 discloses a basic schematic of the present invention. Figure 2 discloses a preferred intake port for the present invention. Figure 3 discloses a preferred embodiment of the present invention. Figures 4a-4c disclose isokinetic separators used in the present invention. Figure 5 discloses an alternative embodiment of the present invention. Figure 6 discloses isokinetic flow model results for the present invention. Figure 7 discloses potential-based fluid flow mathematical simulation results for the aerodynamic sorting of the present invention. Figure 8 discloses a typical deployment scenario of the present invention. DETAILED DESCRIPTION OF THE INVENTION The present invention is to method and apparatus for directing harmful agents onto a bioreceptor surface for use in detecting and classifying chemicals, poisons, particles, vira, and bacteria in fluids, herein collectively referred to as agents. As used herein, the term fluid is intended to comprise, without limitation, any fluid including air and water, or fluids used as intermediates to convey agents to the device from solid objects or surfaces. The present invention incorporates a sensor for biological pathogens and poisons, chemical poisons, including neurotoxins and metabolic poisons, and drug or weapon precursor chemicals in the air or other fluids. The sensor is based on either an aptamer or a quiescent cell treated with fluorescent or luminescent dye to evoke an optical response. For cells, this can be in response to an intracellular ion channel signal, typically, but not exclusively, K+, Na+ or Ca++. The aptamer or sensor cell is selected and/or trained to respond to selected antagonists and ignore others. In a preferred embodiment, the bioreceptor is either an aptamer or a sensor cell that is poikilothermic and sits in the quiescent state in a conventional nutrient gel medium, or on a suitable organic substrate, protected from evaporation by a hydrophobic barrier or otherwise selective barrier or filter, porous to aerosols, particles and spores of a desired diameter and to gases dissolved in the in the atmosphere. A suitable hydrophobic barrier can be formed as a membrane or filter and can be applied at the intake, outlet, or covering the wells of the sensitive region. One suitable material for the hydrophobic barrier is polytetrafluoroethylene (PTFE) film with perforations of approximately 10 μm in diameter. The aptamers or sensor cells chosen and/or trained for this task should have certain properties discussed below. The term trained refers to any method for selecting or controlling the receptors or receptivity internal or external, of that aptamer or cell. These properties: • have minimal changes in sensor characteristics due to temperature, pressure or humidity changes • respond to desired challenges and not respond to other challenges in selected patterns of response/non-response • provide optical (including fluorescent and/or luminescent) indication or readout of cell activation, optionally incorporating both space-dependent and time-dependent signaling characteristics • provide optical (including fluorescent and/or Iuminescent)readout of cell activation including space-dependent and/or time-dependent processing of the readout data as detection and classification variables • possess minimum quiescent metabolic requirements • possess minimum sensitivity to temperature variations • provide response to classes of challenges not previously catalogued and/or for which specific antibodies, DNA sequences or protein sequences may have or have not been determined • be normally-quiescent and poikilothermic (cells) • provide conversion of ionic signal responses (cells) to photons via luminescent or fluorescent means for the purpose of producing a readout of response to viral, bacterial or chemical challenges of selected or broad natures, and • provide means for generating antibodies to collected agents within the apparatus. The biosensor of the present invention may incorporate Aristotelian and/or Non- Aristotelian logic, including multi-valued logic and statistical estimation to provide diagnostic information incorporating resistance to deliberately induced or accidental false alarms. An example of a usable sensor cell is an activatable heterotrophe rendered sensitive to the desired agent by special biological modification methods. Other examples include encysting ameba, epidermal cells, melanocytes, macrophages, phagic yeasts, and certain species of fungi, which in the preferred embodiment are poikilothermic and quiescent until activated. Each sensor may contain a replaceable plate or cartridge for holding the aptamer or sensor cell, with number of containers dependent on the deployed embodiment, such as in a rectangular or hexagonal configuration, with outlet holes for the air or other fluid interspersed among the aptamers or cells in the plate. The biosensor container is generally in the form of a non-spherical optics subsystem and container, with reflective and/or refractive means to concentrate emitted photons to a small region external to the container. Each container terminates in a conventional photocell that collects the emitted photons resulting from activation of the aptamer or cell, either via luminescent response, such as, but not limited to, infused luciferase or aequorin, or via fluorescent response to infrared, ultraviolet or visual light stimulated emission. The collected photons are then converted to electrons by conventional photocell means and processed in a known manner to elicit space-time characteristics of the response. The results, incorporating both space-time characteristics of pattern response to activation of bioreceptors in individual containers, and the pattern of containers activated, are processed via appropriate Aristotelian and non-Aristotelian (probabilistic) logic to yield diagnostic signals. Preferably, the challenging substance is also collected in the medium itself for further analysis. Additionally, the invention includes but is not limited to the use of shutters or trapdoors to control which bioreceptor containers are available to activation at a given time. The invention includes the feasibility of rapid generation of antibodies using the device and captured samples, after exposure. In its most basic form, as shown in figure 1, the present invention provides a means to concentrate and impinge particles, spores, vapors, and gasses from air or another fluid medium onto the bioreceptor. The fluid is taken in either through natural stream flows or via a small fan as shown at 10, and passed through a filter designed to remove large particles from the fluid as shown at 20, which may also incorporate the hydrophobic barrier discussed above. The resulting flow is then introduced into a chamber after optionally being electromagnetically charged and directed. The impingement includes, without limitation, presenting pathogenic organisms, whether or not incorporated in aerosols, and/or toxins and poisons (including "poison gas" agents) suspended in the fluid as aerosols, vapors, gases, solutions, particles or by other means, onto sensitive surfaces of the bioreceptor intended to process the material, as shown at 50. The chamber consists of a series of collectors at one end, interspersed with one or more exit orifices. An optional sinusoidal or otherwise varying electrical field designed to optimally control the motion of the particles within the chamber sets up electrodynamic forces that cause the collectors, which are part of the electrodynamic apparatus, to attract the agents being collected and/or tested. This mechanism is discussed further below. The motion of the particles is generally in the form of a nonlinear vector motion and/or, but not limited to, a wave of increasing amplitude, terminating in impingement on the bioreceptor surface. The remaining components of the fluid exit the chamber, and optionally pass through a finer filter to provide additional collection means. The sensitive structure consists of a plate, volumetric receiver or array or other array of one or more sensitive bioreceptors of the present invention, each of which provides one or more components of the analysis that the entire system is assigned to accomplish. The fluid passage optionally contains a series of designed bends or labyrinth, as shown at 30 whose purpose is to block light from the sensitive materials, as well as providing a portion of the aerodynamic filtering. This is in addition to the conventional mechanical filtering cited above. The aerodynamic and kinetic filtering of 40 consists of one or more of three components. First, the bends in the passage are designed to employ a balance of centrifugal and drag forces to select out particles whose ratio of radius-squared to total mass (i.e. density times radius-cubed times four-thirds Pi) meets desired characteristics, mediated by the deliberately variable airflow in the bent section. Other mathematical functions serving as criteria for forting and selection include Bernoulli forces, and the effects of Reynolds and Froude Numbers as examples. A structure bearing an aerodynamic functional resemblance to the Turbinate bone of vertebrate animals (found elsewhere in Nature as well) is optionally utilized to assure proper randomization of the components, as well as to cause early non-sensitive impingement of particles of undesired effective (i.e. aerodynamic) diameters. In the preferred embodiment the optimum particle size for impingement is matched to the alveolar capture function, described below. Each sensitive detector is optionally covered with a small trapdoor which when closed by electrical or mechanical means seals an associated detector. When open, the trapdoor optionally erects to a critical angle obtuse to the fluid stream, providing further optional selection of components of the stream using laminar or turbulent vortices to further provide discrimination for particle size and density. The optional electromagnetic component of the device consists of a varying electrostatic field with incidental magnetic field optimized to accelerate particles of the appropriate size (surface area) and density onto the sensitive surface. The field either utilizes the induced dipole component of the particles being sorted or selected, or makes use of an electrostatic monopole on the each of the particles, using traditional methods for inducing the electrostatic monopole. The fluid exits from the chamber (i.e., the chamber containing the plate or array and associated components) through holes in the plate or array interspersed among the detectors. The fluid then optionally passes through another light-restricting labyrinth to the exit and optional collection filter as shown at 60. In a preferred embodiment of the present invention, for combining with a sensor, the system has the responsibility of : (1) extracting desired aerosol, vapor, gases and solid particles from the air, (2) eliminating particles of inappropriate size (e.g., dust on one end and smoke on the other), (3) sorting these particles while preserving viability of carried organisms, and (4) concentrating and impinging the desired particles on the sensitive surface of the sensor. The system can be broken down into four subsystems, not precisely corresponding to the above functions. It is optimized for acquisition of particles (aerosol or solid) forming particular mammalian threats, i.e. nominally l-5μm in mean diameter. The first subsystem is concerned with particle acquisition and large-particle sorting. The challenge is that of capturing valid air samples despite wind direction and velocity. For this task the present invention preferably uses an omnidirectional assembly of the NACA flush intake design used in low-speed aircraft, which has the advantage of providing a positive Bernoulli-derived pressure to assure that sampling takes place. This has the further advantage of minimizing rain and small-particle effects, which could have an undesired effect on a sensor package. One such flush intake is diagramed in figure 2 in which fluid such as airflow 210 is taken into NACA intake 200 in plate 220. One such duct is of course partially unidirectional. To act omnidirectionally, the system can use four or more such intakes 300 disposed around a circle, and covered by a hat or cover 310, as shown in figure 3. These intakes 300 have a common (large-diameter) central opening 312, with a smooth transition to a common intake 314. The main motive power for sampling is provided by a small DC-operated axial fan 320 moving approximately 15 L/m of air at very low pressure (pressure to be determined experimentally). This fan may be placed at the entrance or exit of the system. The next subsystem concerns mechanical filtering. The entranceway 316 to these intakes 300 are covered by a mechanical dust filter 318 of high porosity. This filter 318 can be prepared using laser forming techniques and a hydrophobic base, available from Laserfare Inc. of Newport, RI . The design of the filter should be made to exclude particles of minimum diameter >10mm with minimum back-pressure. The mechanical filtering components generally act on the mechanical minimum diameter of a particle. In the region of interest here, the Reynolds Number Re<l and the orientation of an elongated particle is random. However, the primary filtering method to eliminate, for example, dust and smoke, must therefore act on the aerodynamic (actually Stokes) diameter of the particle, and therefore mechanical filtering is insufficient. Note that from elementary geometric considerations derived from close-packing theory, the maximum theoretical porosity of such a filter can be shown to approximate — γ= . Practical porosities of the order of 80% are therefore attainable, provided that a 2V3 nonrandom filter-building process is utilized. A randomly generated filter (such as might be prepared chemically) has much lower porosity. The optimal design is that of the inversion of a hexagonal lattice in the X and Y dimensions (normal to the airflow) and a slightly bent tube in the Z direction. At the time of manufacturing, the laser-cut filters can be used to prepare molds for casting multiple copies, to reduce costs. Because mechanical filtering alone is insufficient for desired system, the next subsystem concerns aerodynamic isokinetic filtering. Selection of particles having the desired aerodynamic diameters (as opposed to minimum diameters) is aerodynamic in nature. Figures 4a-4c illustrate the general dam mechanism as element 400a, 400b, and 400c. In figure 3, the fluid passes labyrinth 330 to pass over dams 340 for isokinetic filtering to collect particles of interest for impingement on the sensors of sensor array packages 350, which are preferably replaceable. Remaining fluid passes exit labyrinth 360 to exit/collection filter 370 and fan 320. The design of the isokinetic filter is somewhat counterintuitive, involving dams 400a, 400b, or 400c at angles of attack ( ) in the deep-stall region, i.e., greater than 90 ° in the preferred embodiment, but is borne out by research, analysis and computer simulation. In the regime of interest, aerosol particles having approximately the same density can be sorted by mean aerodynamic diameter quite efficiently. For the regions of interest, the diagram of figures 4a-4c are illustrative, and preliminary calculations have established the notional angle of attack of the aerodynamic filter. In figure 4a, orifice 410a acquires particles of interest for impingement onto a surface. It is integrated into the dam 400a and is located on a backside (i.e., downstream side) thereof at a location appropriate for the desired isokinetic sorting. Figures 4b and 4c disclose similar embodiments. In figure 4b, a collection tube 420 is used to position an orifice 410b and in figure 4c, orifice 410c is positioned on the base 430 adjacent to the dam 400c. The common aspect of all of these embodiments is that the orifice is at the downstream side of the dam or otherwise shielded from direct flow by the dam. Locating the orifice on the dam, on a separate collection tube, or on the base adjacent to the dam, but on the downstream side thereof, facilitates the isokinetic sorting. The ratio of stream velocity to orifice velocity, the angle of the dam, and the position and size of the orifice regulate the distribution of particles that enter the orifice. This is demonstrated in the simulation. Although orifices 410a and 410c will generally be fixed, orifice 410b can optionally be easily repositioned by making collection tube 420 movable. For example, although there is no functional difference between an orifice 410a in the dam 400a and an orifice 410b on a collection tube 420 positioned to be shielded by the dam 400b from the prevailing flow vector, the angle of collecting tube 420 with respect to the base (i.e., X- axis) is also a solution variable. Sorting can be varied based on repositioning of the collection tube 420. Dam 400c can also have utility as a trap door to cover a sensor. Basically, as shown in figure 7, the dam 700 sets up a local vortex 710 which sorts the particles isokinetically, by density and aerodynamic diameter. The underlying theory is also discussed below, with preliminary analysis of the phenomenon, both extrapolated from experimental data and supported by a first-order simulation. The final subsystem concerns impaction. After some analysis, the present inventors have concluded that the conventional aerodynamic impaction methodologies exhibited in commercial off-the-shelf viable-particle impactors are satisfactory and probably optimal for the present application. An existing impaction chamber design, such as those available from Anderson Instruments, 500 Technology Court, Smyrna, GA 30082, USA, is therefore usable for the present invention. Figure 5 illustrates an alternate embodiment of the invention in which such an impaction chamber 507 is employed. In this embodiment, large particles and rain are excluded by input rain and sun hat 501 and large particle filter 502. Fluid then passes through NACA fluid dynamic intake orifices 503 to a collector and second filter 504 to an intake labyrinth and venturi 505 that directs the fluid over fluid dynamic dam 506 which acts as an inertial filter to obtain particles of interest at an orifice at the rear. Particles of interest are directed by inertial impactor 507 (which can optionally be replaced with the electrodynamic impaction discussed above) over the open trap doors of sensors 508 which are optionally replaceable via drawer 509. The remaining fluid is collected in chamber 510, passed through exit labyrinth 511 to fan 512 to exit collector and filter 513. The apparent particle density of an aerosol is generally much smaller than the actual density, with coefficients of sphericity (ratio of surface area of an equivalent sphere to actual surface area (A/A o )). This means that for organic aerosols the apparent density is uniformly less than unity, and in the range where Stokes' Law does not apply experimentally very well. For the region of interest it amounts to a correction to Stokes' Law, and the inventors have chosen to use experimental data as guidance. Below, it is noted that preliminary simulation results using the assumptions underlying Stokes' Law are quantitatively consistent with the extrapolated experimental data. Robinson (see Comm. Pure Applied Math, 9,69) demonstrated that in these cases gravitational and inertial collection efficiencies are additive. From the point of view of alveolar deposition (which is most critical from an infection viewpoint, and therefore defines both threat and measurement),the most recent data from Lippmann (see Lippmann, M. "Regional Deposition of Particles in the Human Respiratory Tract", in Handbook of Physiology, American Physiological Society, Bethesda, MD 1977) suggests that for both oral and nasal alveolar deposition the optimum aerodynamic diameter is of the order of 1-5 μm, with tails of about .7-7 μm at the 15% efficiency level. Without belaboring the derivation, from Stokes' Law and the assumptions of low Reynolds Number it can be shown that gravitational settling is fairly independent of wind velocity (including turbulence) and is given by 8 * [( 4/ 3)πr 3 lγ γα Re φ = - — where Re is the Reynolds Number, which by πη definition = 2rγ a V / η yielding the velocity V, and where γ = density of the particle γ α = density of the air g = gravitational acceleration η = viscosity of the air There are better approximations based on empirical data, but for this analysis and design purposes they can be ignored unless it is required to accurately measure particle density and mean radius. Data from Watson (see Amer. Ind. Hyg. Ass. Quarterly, 3,29) is shown in figure 6, which has been nondimensionlized (appropriate in the region of interest) has been extrapolated by the inventors to cover the expected particle size range (p eff « 1), indicating how the sorting mechanism will select out particles of the appropriate size. These data are fortunately nondimensional, and confirm the present design and analysis. Most importantly, smoke particles, having effective diameters well under .5 μm will be rejected. For the aerodynamic sorting, the entire air-handling problem is properly modeled by the Navier-Stokes formulation. supplemented by the Equation of Continuity (incompressible flow in this case) and the thermodynamic relationship (generally adiabatic flow with γ « 1.5, but also negligible in this instance). Using a potential-based fluid flow simulator, available from the U. Mich. Department of Aerospace Engineering, a simple model of an appropriate obstruction to uniform flow was modeled at an appropriate angle. The results are illustrated in figure 7. Assumptions included incompressibility, and irrotational laminar flow. These assumptions are appropriate to the scenario including the prevailing Reynolds numbers («1). A number of runs were made, varying appropriate parameters (i.e., ratio of source to sink rates and angle of incidence), to demonstrate the aerodynamic filtering mechanism. The airfoil angle of incidence in the pictured simulation was 210°. The non- dimensional input flow rate of the pictured simulation run was 1 sec "1 , and the sink rate was located at a notional fluid sink of 2 sec "1 at the ventral center of an arbitrarily thin barrier. Other ratios of source to sink rate (1 : 1 to 1:3) provided similar results. The streamlines are shown. The equipotential lines are of course orthogonal to the streamlines, and reveal no surprises. Our analysis has shown that the optimal incidence angle may vary from the incidence angle shown, including angles in the laminar, turbulent and stall regions, depending on the selection criteria, densities, velocities, and Reynolds numbers. Furthermore the collection orifice, while not directly in a streamline, may be partially so, located on the barrier, or on the substrate of the barrier, again depending on the conditions of design and operation. The class of inlets used for optimum pressure recovery, referred to as "NACA" or NACA-type" are generally unsuitable to this solution. From these results, it is fairly obvious under the conditions in which Stokes' Law is valid (these being the case) that, to the first order, a particle of density p with a mean radius of r embedded in a much lighter fluid (p f «p p ) having a curved path with radius r a and a flow velocity of v a will have a terminal velocity component v p normal to the fluid streamlines (neglecting gravity, which can be vectorially added later) of: v _ 4prV a 2 _ pr^ P 18μr a r a which provides square-law sorting by particle size. Varying the incidence angle of course changes the flow and the local curvature of the streamlines, and the present invention anticipates an incidence angle of 180<α<270 (see simulation graphic) for particle sorting, and, from the simulation, for the stated conditions, αs210 degrees seems optimal to provide the greatest dispersion of particle paths. Thus there is an approximate agreement between the extrapolated empirical data and the simulation. The exact dam and entry angles can vary from those disclosed herein. (Note that 120° in the empirical diagram corresponds geometrically to an incidence angle of 210°.) This computational experiment merely serves (1) to demonstrate qualitatively the measured filtering effects and (2) provide a basis for detailed analytical simulation in the course of the project to optimize the aerodynamic sorting phase of the preferred embodiment of the present invention. In use, the sensors of the present invention can be configured to be hand emplaceable, as shown in figure 8, or wearable (not shown). In a preferred embodiment, sensor units 810 are deployed and are linked, such as by radio links 820, to a central readout and decision center 830. If the sensor units 810 are deployed with military units or near populations, a local alarm 840 can be used to warn personnel. An alternative deployment involves groups of individuals each carrying a wearable sensor sensitized for and optimized for a smaller class of agents. The aggregate wearable sensors in this group communicate locally among each other via low-powered radio signals, forming a self-organizing redundant network, to achieve a combination of selectivity and breadth of response. The aggregate results are then optionally communicated to the central readout and decision center. The central readout and decision center 830 can (1) provide detection and classification of the agent, depending on the strength and signal time-characterizations reported by each well or container, and the pattern of responses among all the containers within the apparatus, (2) minimize both false negatives and false positives in the decision (3) optionally deal with uncertainty or unanticipated patterns of response — including spoofing and obscuration — according to probabilistic rules, (4) compose an annunciation and diagnostic report message, and (5) transmit that message or annunciation by conventional means. The underlying software algorithms were separately developed by one of the present inventors (Leibholz) and the software is protected as a copyright. When deployed using cells, harmful agent exposure results in timed activation of cells and initiation of phagocytic activity in less that one minute from exposure, beginning at 100 μsec and reaching peak value within one (to ten) seconds of binding. As cell activation is usually coordinated, in part, by a large amplitude signal, activation of a cell can be annunciated with a suitable luminescent (e.g., aequorin) or fluorescent (e.g., fura2) intracellular calcium reporter. Aptamer-based bioreceptors: The general process of the present invention for using aptamer-based bioreceptors is analogous to that of the use of genetic algorithms to construct the desired sequences. The affinity of a putative aptamer to a target is determined by geometric as well as chemical considerations, being a mixture of folding properties, multipole electrostatic and van der Waals forces, generally both covalent and noncovalent bonds. The photonic conversion reporter process is a result of polarization changes rather than Ca++ channel changes in the sensor biomolecules. A sensor aptamer of the present invention can be produced and deployed, for example, by the following method: A. Develop targets of interest (e.g., JCS list agents, bacteria, pollens, viruses, prions, metabolic toxins, other pathogens, narcotic precursors and by-products, et cetera) by using infective and replication processes known in the literature; B. Collect T-cell and B-cell populations where appropriate; C. Prepare a large number of pseudorandom sequences of nucleic acids; D. Mix them with the targets in solution phase; E. Discard the unbound or weakly bound molecules, e.g., via electrophoretic or chromatographic methods; F. Continue process of collecting adhering candidates, further modification through randomization and resorting proceeds until a desired degree of affinity is obtained; G. Attach the photonic reporter molecules; H. Attach the combined molecules to the substrate, typically a decelularized biomembrane; and I. Test and repeat. Cell-based bioreceptors: A sensor cell of the present invention can be produced and deployed, for example, by the following method: A. Immunize appropriate cells with agent of interest (e.g., JCS list agents, bacteria, pollens, viruses, prions, metabolic toxins, other pathogens, narcotic precursors and by- products, et cetera); B. Collect T-cell and B-cell populations; C. Develop hybridomas for suitable positive T-cells and B-cells; D. Isolate mRNA for B-cell surface antibodies of interest (i.e., using receptors having various Kd for immunogen); these receptors may be for any selected quorum, including but not limited to surface proteins where applicable; E. Isolate cytokine receptor of a sensor cell; F. Isolate mRNA for cytokine receptor of sensor cell; G. Immortalize sensor cell with HTLV-1 or myeloma fusion; H. Transform the sensor cell to express photoprotein for calcium (e.g., aequorin, obelin, et cetera) or free radicals; I. Transform sensor cell with multiple chimera receptors (e.g., macrophage intra- membrane and cytoplasmic domains of cytokine receptor of the sensor cell and extracellular and intra-membrane ligand binding domains of educated B-cell); J. Culture cells in multi-well culture device wherein each well contains hydrogel, suitable matrix for cells, and culture media including lumiphore; K. Cells may also be labeled with fluorescent and/or absorptive probes for materials of interest; L. Agents of concern are to be presented to the cells via the collector structure described above or other suitable means; M. On binding of ligand, cells activate and emit photons from calcium-dependent luminescence; N. Pattern of luminescent wells versus controls is characteristic of activating agent; O. Pattern is decoded by on-board logic and annunciated through on-board display and/or transmitted to remote receiver and decoder; P. After annunciation, cells can be retrieved and residual bodies probed for identity of activating agent; Q. General metabolic toxins will also give luminescent output, which will be classifiable due to broad array response; R. Unknowns will be identifiable through activation of controls and suitable general class-identifier cells; S. T-cell and B-cell clones produced will be available for subsequent analysis of residual bodies; T. New sensors can be readily configured and validated for shipment, and subsequent field deployment, with a target field life of 1 ,200 hours on-station; U. Use of lower Kd chimera receptors will afford means of both amplitude/dose discrimination and altered physiognomy of ligand; V. Use of multiple chimeric receptors affords equivalence of pooled monoclonal antibodies. This invention therefore provides for sensing biological, chemical or radiation challenges to humans, animals, and plants even when that challenge has not previously been catalogued, isolated and/or analyzed. It also can be used to reliably detect biological, chemical or radiation challenges to humans, animals and plants despite the presence of natural or deliberate mutations, and/or conformers, enantiomers and/or chimeric agents. A technology that is useful with the present invention is the control of nuclear envelope breakdown (NEB) in the sensor cells, both to speed up cell production prior to deployment and to enhance cell use after deployment. From earlier published documents, it is known that cells produce and probably require a discrete calcium signal prior to nuclear envelope breakdown. This calcium signal is expressed from intracellular endomembranes acting as the sink and source for the calcium signal. From earlier published documents, the calcium-uptake activity of ATP-dependent calcium pump enzyme of the endoplasmic reticulum (ER) is required for cell division.. The calcium signal is expressed as a discrete space-time pattern. From earlier published documents, for inhibition of cell division, disruption of the space-time pattern of the pre-nuclear-envelope-breakdown (pre-NEB) calcium signal is employed which results in cessation of nuclear envelope breakdown and thus cell division. From earlier published documents, the enzymes essential to produce cell division are present as part of the peri-nuclear endomembranes. A selective, properly timed inhibition of any of the enzymes needed to produce this cell division, particularly leukotriene B4 (e.g., phospholipase A2, 5-lipoxygenase, leukotriene A4 hydrolase) results in blocking of NEB and subsequent cell divisions. From earlier published documents, the production of leukotriene B4 is part of an integrated network of enzymes spanning the 5-lipoxygenase, glutathione reductase/glutathione S-transferase-leukotriene c4 synthase, pentose phosphate and glycolytic pathways, and likely involves feedback with purine, pyrimidine and amino acid biosynthesis. Thus, disruption of the space-time pattern of the pre-NEB calcium signal either by superimposing a random or other pattern, or disrupting biosynthesis of leukotriene B4 will result in blocking of nuclear envelope breakdown and cell division. For initiation or enhancement of the probability of cell division, the space-time pattern of the pre-NEB calcium signal is mimicked or enhanced, using open-loop chemical, photonic or electrical means, or using closed-loop augmentation of the natural signals. Such a disruption, properly applied in space-time, is can thus be used to stop cell division (e.g., to terminate a cancer, et cetera). This disruption may take the form of continuous (DC) or random signals, as well as periodic signals of appropriate frequency, waveform and amplitude. Similarly, expression of such a space-time patterned signal as normally occurs in normal cells, maybe used to restore the cell division process in cells that have stopped cell division (e.g., aplastic anemia, et cetera) or in which controlled cell division needs to be restored (e.g., regeneration of burned tissues, et cetera). This signal may be generated in open-loop or closed-loop fashion, either creating the desired response or enhancing it.

Claims: We Claim: 1. A method of detecting and classifying chemicals, particles, vira, and bacteria in fluids, comprising: selecting and/or training a bioreceptor selected from the group consisting of aptamers and poikilothermic and quiescent cells to be sensitive to a desired agent; treating said bioreceptor with fluorescent or luminescent dye to evoke an optical response in the presence of said agent; and detecting photons emitted by said bioreceptor in the presence of said agent. 2. The method of claim 1, wherein said bioreceptor is a cell selected from the group consisting of activatable heterotrophe, encysting ameba, epidermal cells, melanocytes, macrophages, phagic yeasts, and fungi. 3. The method of claim 2, wherein said cell is treated with infused luciferase, aequorin or fura2. 4. The method of claim 2, wherein said optical response is an intracellular ion channel signal selected from K+, Na+ or Ca++. 5. The method of claim 1, further comprising: capturing a fluid sample by drawing it through an intake; mechanically and/or aerodynamically filtering out particles > lOμm; using a dam to set up a local vortex to isokinetically sort particles by density and aerodynamic diameter; and selecting particles within a selected size-range for impaction on a surface of said bioreceptor. 6. The method of claim 1, further comprising: capturing a fluid sample; filtering out particles > 1 Oμm; isokinetically sorting particles by density and aerodynamic diameter; and selecting particles within a selected size-range for impaction on a surface of said bioreceptor. 7. The method of claim 6, wherein said selected size range is approximately 1-5 μm. 8. The method of claim 6, wherein said fluid is air. 9. The method of claim 6, further comprising collection of impinged agents that cause activation. 10. A method of biosensing an agent by collecting, sorting, concentrating and impinging particles on a surface of a bioreceptor, comprising: capturing a fluid sample by drawing it through an intake; mechanically and/or aerodynamically filtering out particles > lOμm; using a dam to set up a local vortex to isokinetically sort particles by density and aerodynamic diameter; and selecting particles within a selected size-range of said agent for impaction on the surface of the bioreceptor, wherein said bioreceptor is selected from the group consisting of aptamers and cells that are poikilothermic and quiescent. 11. The method of claim 10, wherein said bioreceptor is a cell selected from the group consisting of activatable heterotrophe, encysting ameba, epidermal cells, melanocytes, macrophages, phagic yeasts, and fungi. 12. The method of claim 11, wherein said cell is rendered sensitive to said agent by special training methods. 13. The method of claim 10, further comprising providing a photocell, wherein said bioreceptor is treated with fluorescent or luminescent dye to evoke an optical response in the presence of said agent so as to be detected by said photocell. 14. The method of claim 10, wherein said selected size range is .7-7 μm. 15. The method of claim 10, wherein said selected size range is 1-5 μm. 16. The method of claim 10, wherein said dam has an angle of attack >90 degrees with respect to said fluid. 17. The method of claim 10, wherein said dam has an angle of attack between 180 and 270 degrees with respect to said fluid. 18. The method of claim 11 , wherein said dam has an angle of attack of 210 degrees with respect to said fluid. 19. The method of claim 11 , wherein said fluid is air. 20. An apparatus for biosensing an agent by collecting, sorting, concentrating and impinging particles on a surface of a bioreceptor, comprising: a fluid intake; a mechanical filter with an effective mesh size of 10 μm; and a fluid flow conduit having at least one dam means to set up a local vortex to isokinetically sort particles by density and aerodynamic diameter, wherein said dam means includes means for selecting particles within a selected size-range for impaction on a surface, wherein said sensor cell surface contains a sensor cell that is poikilothermic and quiescent. 21. The apparatus of claim 20, wherein said bioreceptor is a cell selected from the group consisting of activatable heterotrophe, encysting ameba, epidermal cells, melanocytes, macrophages, phagic yeasts, and fungi. 22. The apparatus of claim 21 , wherein said cell is of a type that has been rendered sensitive to the desired agent by special training methods so as to evoke an intracellular ion channel signal. 23. The apparatus of claim 20, further comprising a photocell, wherein said bioreceptor is of a type that has been treated with fluorescent or luminescent dye so as to evoke an optical response in the presence of said agent to be detected by said photocell. 24. The apparatus of claim 20, wherein said fluid intake is an omnidirectional array of NACA flush intakes. 25. The apparatus of claim 20, wherein said mechanical filter includes a labyrinth element to exclude light. 26. The apparatus of Claim 20 incorporating an element formed by laser forming or laser-formed molding techniques. 27. The apparatus of claim 20, wherein said selected size range is .7-7 μm. 28. The apparatus of claim 20, wherein said selected size range is 1-5 μm. 29. The apparatus of claim 20, wherein said dam means has an angle of attack >90 degrees with respect to said fluid. 30. The apparatus of claim 20, wherein said dam means has an angle of attack between 180 and 270 degrees with respect to said fluid. 31. The apparatus of claim 20, wherein said dam means has an angle of attack of 210 degrees with respect to said fluid. 32. The apparatus of claim 20, further comprising a fan positioned to cause fluid flow through said fluid intake. 33. The apparatus of claim 20, wherein said dam means includes an upstream dam element and said means for selecting particles is an orifice on a downstream side of said dam element, wherein said orifice is located at a position selected from the group comprising a downstream side of said dam element, an end of a collection tube located downstream of said dam element, and a base adjacent a downstream side of said dam element. 34. The apparatus of claim 20, further comprising an interconnection of a plurality of biosensing apparatuses via a central readout and decision system. 35. The apparatus of claim 20, further comprising a means for generating antibodies to the sensed agent, within the apparatus or an adjunct device. 36. The apparatus of claim 20, configured into a network of sensors, with aggregate capabilities greater than those of any single sensor at one general location. The apparatus of claim 36, wherein the network of sensors includes wearable sensors.

Inventors: LEIBHOLZ STEPHEN, MANNIELLO JOHN, LEIBHOLZ, STEPHEN, MANNIELLO, JOHN
Applicants: CHESAPEAKE TECHLABS INC, LEIBHOLZ STEPHEN, MANNIELLO JOHN, CHESAPEAKE TECHLABS, INC., LEIBHOLZ, STEPHEN, MANNIELLO, JOHN
Application year: 2001
Main classification: G01N33
IPC codes: G01N1, G01N15, G01N33