WO2003104810A1

WO2003104810A1 - Biological warfare sensor using molecularly imprinted polymers

Info

Publication number: WO2003104810A1
Application number: PCT/US2003/018242
Authority: WO
Inventors: Jay A. Glasel; Carl J. Freeman
Original assignee: Sensor Research And Development Corporation
Priority date: 2002-06-07
Filing date: 2003-06-09
Publication date: 2003-12-18
Also published as: AU2003247518A1

Abstract

A sensor (10) for sensing biological warfare agents including a transducer and a molecularly imprinted polymer applied to a surface of the transducer. The molecularly imprinted polymer has at least one site capable of selectively binding a biological warfare agent formed therein. In one embodiment, the transducer can be a quartz microbalance including a quartz chip (14) to which the molecularly imprinted polymer (12) is applied, an oscillator (16), and a controller (18). The oscillator causes the quartz crystal chip to undergo oscillations of increasing amplitude so as to rupture any bonds between biological warfare agents and the molecularly imprinted polymer. The controller is capable of detecting such rupture events.

Description

BIOLOGICAL WARFARE SENSOR USING MOLECULARLY IMPRINTED POLYMERS

BACKGROUND OF THE INVENTION

[0001] This invention relates generally to sensing biological warfare agents and more particularly to a sensor that incorporates an organic polymer-based biological warfare agent detector combined with proven, highly sensitive sensor technology.

[0002] The distribution of biological warfare aerosols is highly effected by atmospheric conditions such as rain and wind. Therefore for any effective use in detecting the severity of a biological warfare attack over a wide geographic area, a large number of biosensors spread over the area will be needed. This means that field-operable biosensors must be simple, sensitive, accurate, and reproducible.

[0003] As recently reviewed (Paddle, B. M. (1996) Biosensors for chemical and biological agents of defense interest Biosens Bioelectron 11 1079-113; and Hobson, N. S., et al. (1996) Microbial detection Biosens Bioelectron 11 455-77), almost the entire known range of physical and biological approaches has been applied to developing biosensors for biological warfare agents. Despite this enormous effort, relatively few agents can yet be measured reliably by commercially available equipment.

[0004] Useful biological warfare biosensors must be highly selective as well as highly sensitive. In current field-operable biosensors this requirement is satisfied by incorporating one or more biological-derived elements in the detector part of the biosensor. In most current sensitive biosensors, the biological-derived elements are antibodies that selectively bind to specific biological warfare agents. In an antibody-dependent biosensor, the biological warfare agent-antibody binding event is then converted to a signal by one of many available transducers, which can monitor that event. [0005] But antibody-containing biosensors suffer from the problems that antibody isolation and production is expensive, efficient binding of antibodies to transducers is often difficult and, because they are proteins, antibodies may be destroyed by dehydration, bacterial action, moderately high temperatures, and by a variety of other environmental conditions.

[0006] Accordingly, there is a need for biological warfare sensors that do not rely on biomolecules such as antibodies.

SUMMARY OF THE INVENTION

[0007] The present invention provides an improved biosensor that combines development of synthetic polymers that selectively bind biological warfare agents with proven, highly sensitive transducer instrumentation. One unique aspect of this biosensor is the method of detection of biological warfare agents. The invention utilizes molecularly imprinted polymers (MIPs) that selectively bind intact biological warfare agents on the basis of the agents' characteristic three-dimensional "footprints" that are permanently imprinted in the polymers. The MIPs replace biomolecules such as antibodies as the biosensor components that bind biological warfare agents selectively and strongly. This reduces production costs of field-operable biological warfare biosensors greatly while at the same time increases the biosensors' stability in the face of environmental challenges.

[0008] In one embodiment, the present invention is a polymer-based array biosensor that has a limit of detection (LOD) < 100 bacterial or viral biological warfare organisms when applied in a microliter-sized sample. The present invention is able to identify known biological warfare agents and to distinguish pathogenic biological warfare agents from non-dangerous stimulants. The detector part of the present invention is based on the specific binding of each agent to a polymer molecularly imprinted with a negative "image" of the agent's three-dimensional structure and applied to the surface of a crystal quartz (CQ) chip as a thin film. The transducer is based on forced oscillations of variable but defined magnitude driven by an alternating current piezoelectric effect. The transducer is able to differentially identify the species of the polymer-bound bacterial and/or viral agents when they are shaken loose by the forced oscillations of the CQ chip at an amplitude of oscillation characteristic of the binding strength of each biological warfare agent to its imprinted polymer.

[0009] The present invention combines thin film technology with off the shelf commercial transducer components and instrumentation to provide a small, rugged biosensor that can be mass-produced. This technology can be applied to detection of a single type of biological warfare agent, or used as arrays of miniaturized MIPs detectors on CQ chips. The arrays will be sensitive to the range of known biological warfare agents and result in a biosensor that can achieve rapid and accurate simultaneous field detection and identification of a wide variety of aerosolized biological warfare agents.

[0010] The present invention and its advantages over the prior art will be more readily understood upon reading the following detailed description and the appended claims with reference to the accompanying drawings.

DESCRIPTION OF THE DRAWINGS

[0011] The subject matter that is regarded as the invention is particularly pointed out and distinctly claimed in the concluding part of the specification. The invention, however, may be best understood by reference to the following description taken in conjunction with the accompanying drawing figures in which:

[0012] Figure 1 is a schematic diagram of one embodiment of a biological warfare sensor.

[0013] Figure 2 illustrates the step of arranging monomers and a template organism for the preparation of a molecularly imprinted polymer film. [0014] Figure 3 illustrates the step of polymerization of the cross-linking monomers for the preparation of a molecularly imprinted polymer film.

[0015] Figure 4 illustrates the step of extracting the template organism for the preparation of a molecularly imprinted polymer film.

[0016] Figure 5 is a schematic diagram of the principle of operation for the biological warfare sensor of Figure 1.

DETAILED DESCRIPTION OF THE INVENTION

[0017] Referring to the drawings wherein identical reference numerals denote the same elements throughout the various views, Figure 1 shows one embodiment of a biological warfare sensor 10. The sensor 10 includes a thin film 12 of a molecularly imprinted polymer (MIP) applied to the surface of a transducer. The MIP film 12 is imprinted with the negative "image" of the three-dimensional structure of at least one biological warfare agent in a manner described below in more detail. In the embodiment of Figure 1 , the transducer is a quartz crystal microbalance (QCM) that includes a crystal quartz (CQ) chip 14 (to which the MIP film 12 is applied), an oscillator 16, and a controller 18. The oscillator 16 produces oscillations in the CQ chip 14, which typically has electrodes deposited thereon. The controller 18 provides power to the oscillator 16 and monitors the frequency of the chip oscillation so as to detect rupture events (described below).

[0018] In the field of molecular analysis, the development and use of molecularly imprinted adsorbents has recently undergone a rapidly expanding role (Andersson, L. I. (2000) Molecular imprinting: developments and applications in the analytical chemistry field J Chromatogr B Biomed Sci Appl 745 3-13; and Andersson, L. I. (2000) Molecular imprinting for drug bioanalysis. A review on the application of imprinted polymers to solid-phase extraction and binding assay J Chromatogr B Biomed Sci Appl 739 163-73) and includes the development of MIPs membranes of the type suitable for the present invention (Vlatakis, G., et al. (1993) Drug assay using antibody mimics made by molecular imprinting Nature 361 645-7). The binding forces between biological warfare analytes and MIPs polymers have been found to be comparable to those between the analytes and comparably selective antibodies (Vlatakis, G._; et al. (1993) Drug assay using antibody mimics made by molecular imprinting Nature 361 645-7). Since the binding of the biological warfare agents to polymers in MIP production doesn't depend on their biological activity, making these imprinted polymers will be simplified by the use of biological warfare agents inactivated by radiation.

[0019] The MIPs are preferably synthetic polymers obtained by polymerizing monomers with a cross-linker in the presence of a template organism (in the case of the present invention, the biological warfare agent or agents to be detected). Referring now to Figures 2-4, the preparation of a MIP film 12 first involves arranging monomers 20 with a template organism or molecule 22 of the biological warfare agent of interest (Figure 2). Next is the polymerization of the functional and cross-linking monomers 20 in the presence of the template 22 (Figure 3). After polymerization, the template 22 is removed by washing or other methods, leaving a site 24 capable of selectively binding the biological warfare agent selectively (Figure 4). The imprinted site 24 represents a negative "image" of a biological warfare agent's three-dimensional structure. The principle behind the binding of the agent to its MIP is the same as for antigen-antibody binding: the three dimensional shape of the target is recognized with high selectivity by the complementary shape of the binding site, and the target is captured and bound by the actions of short-range intermolecular forces acting between the target and its MIP. Contrasted with antibody and other protein absorbents, MIPs are more stable under changing environmental conditions. The strength of the forces between targets and MIPs have been shown to be the same order of magnitude as forces between antigens and antibodies.

[0020] The development and use of imprinted adsorbents has a long history especially familiar to workers in the area of binding of pharmacological agonists to their cellular receptors (Beckett, A. H. and Anderson, P. (1957) A Method for the Determination of the Configuration of Organic Molecules using "Stereoselective Adsorbents" Nature 179 1074-1075; Beckett, A. H. and Anderson, P. (1959) "Footprints" in Adsorbents J. Pharm. Pharmacol. 11 258- 260T; and Beckett, A. H. and Anderson, P. (1960) The Determination of the Relative Configuration of Morphine, Levorphanol and Laevo-Phenazocine by Stereoselective Adsorbents J. Pharm. Pharmacol. 12 228-236T). It has been shown that MIPs selective enough to stereospecifically select small organic molecule optical enantiomers (Beckett, A. H. and Anderson, P. (1960) The Determination of the Relative Configuration of Morphine, Levorphanol and Laevo-Phenazocine by Stereoselective Adsorbents J. Pharm. Pharmacol. 12 228-236T) can be produced. Suitable polymers used for the MIP film 12 include polymers formed as membranes by polymerization of a solution of methacrylic acid and ethylene glycol dimethacrylate monomers on a quartz crystal surface in the presence of the biological warfare agent simulant, B. Subtilis spores.

[0021] The detection of biological warfare agents bound to MIPs is based on an adaptation of a recently reported version of quartz crystal microbalance (QCM) technology called "rupture event detection" (RED) (Cooper, M. A., et al. (2001) Direct and sensitive detection of a human virus by rupture event scanning Nat Biotechnol 19 833-7). In this reported RED technique, biological warfare agents bound to selective anti-agent antibodies attached to the surface of the QCM are forced, by an increasing alternating voltage applied to the quartz crystal (the converse piezoelectric effect), to undergo shearing oscillations of increasing amplitude. At an oscillation amplitude specific for a biological warfare agent-antibody binding, the agent-antibody bond suddenly ruptures producing a micro-acoustic event detectable by its frequency effects on the QC oscillation. The forces to which bound biological warfare agents are subjected by the shearing oscillations are on the order of 10⁶ times that of gravity. This results in a much higher sensitivity to detection for the rupture event sensor in comparison with, for example, cantilever microbalances that bend due to the action of gravity alone. Thus, a rupture event sensor using anti-virus antibodies has been shown to yield LODs for the virus approaching 10 infective units in a 1 microliter sample (Cooper, M. A., et al. (2001) Direct and sensitive detection of a human virus by rupture event scanning Nat Biotechnol 19 833-7; and Dultsev, F. N., et al. (2000) "Hearing" bond breakage. Measurement of bond rupture forces using a quartz crystal microbalance Langmuir 16 5036-5040).

[0022] The transducer in the biosensor 10 can be assembled from standard electronic parts as per the previously published results of others (Cooper, M. A., et al. (2001) Direct and sensitive detection of a human virus by rupture event scanning Nat Biotechnol 19 833-7; and Dultsev, F. N., et al. (2000) "Hearing" bond breakage. Measurement of bond rupture forces using a quartz crystal microbalance Langmuir 16 5036-5040) and can be modified as needed. Commercial components can be used in the assembly.

[0023] The present invention utilizes the rupture signal method, but employs selective MIPs for the detector instead of selective biomolecules such as antibodies. Turning now to Figure 5, the operation of the sensor 10 is discussed. The MIP film 12 is deposited onto CQ chip (not shown in detail in Figure 5), which transduces the rupture's acoustic signal when the biological warfare agent is shaken loose from the surface of MIP film 12. Detection can be based on simultaneous rupture events or sequential rupture events. When a biological warfare agent 26 binds to an imprinted site 24 on the MIP film 12, the CQ chip is forced to undergo shearing oscillations of increasing amplitude. At an oscillation amplitude specific for the binding of the biological warfare agent 26 to the MIP film 12, the agent-MIP bond suddenly ruptures producing a micro-acoustic event detectable by its frequency effects on the QC oscillation. Thus, the sensor 10 is able to differentially identify the species of the biological warfare agent 26 when it is shaken loose by the forced oscillations of the CQ chip at an amplitude of oscillation characteristic of the binding strength of the biological warfare agent 26 to the imprinted site 24. [0024] In addition to higher sensitivity than any currently available biosensor, the present invention has advantages over any device incorporating antibodies or other biological components as detectors:

• MIPs detectors may be mass-produced at very low cost

• The synthetic polymers in MIPs detectors are much more resistant to environmental biological and chemical degradation than detectors based on biological components

• The synthetic polymers in MIPs have high tolerances to mechanical and thermal stress and have excellent storage stability

• Manufacturers can potentially produce combination devices incorporating MIPs sensitive to chemical warfare agents by molecularly imprinting polymers with CW molecular images along with those with biological warfare agent images

• MIPs detectors will basically be self-cleaning ones. Since the signal the present invention produces is based on detection of removal of the adsorbed agent from the MIPs surface, it should prove feasible to renew the surface after each measurement event

[0025] While specific embodiments of the present invention have been described, it will be apparent to those skilled in the art that various modifications thereto can be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A sensor for sensing biological warfare agents, said sensor comprising: a transducer; and a molecularly imprinted polymer applied to a surface of said transducer, said molecularly imprinted polymer having at least one site capable of selectively binding a biological warfare agent formed therein.

2. The sensor of claim 1 wherein said site defines a negative image of a three-dimensional structure of said biological warfare agent.

3. The sensor of claim 1 wherein said transducer includes a quartz crystal chip, said molecularly imprinted polymer being applied to a surface of said quartz crystal chip.

4. The sensor of claim 3 further comprising an oscillator connected to said quartz crystal chip.

5. The sensor of claim 3 further comprising means for causing said quartz crystal chip to undergo oscillations of increasing amplitude so as to rupture a bond between a biological warfare agent and said molecularly imprinted polymer.

6. The sensor of claim 5 further comprising means for detecting rupture of a bond between a biological warfare agent and said molecularly imprinted polymer.

7. The sensor of claim 1 wherein said transducer includes a quartz crystal microbalance.

8. A sensor for sensing biological warfare agents, said sensor comprising: a transducer including a quartz crystal chip; and a molecularly imprinted polymer film applied to a surface of said crystal quartz chip, said molecularly imprinted polymer film having at least one site capable of selectively binding a biological warfare agent formed therein.

9. The sensor of claim 8 wherein said site defines a negative image of a three-dimensional structure of said biological warfare agent.

10. The sensor of claim 8 further comprising an oscillator connected to said quartz crystal chip.

11. The sensor of claim 8 further comprising means for causing said quartz crystal chip to undergo oscillations of increasing amplitude so as to rupture a bond between a biological warfare agent and said molecularly imprinted polymer film.

12. The sensor of claim 11 further comprising means for detecting rupture of a bond between a biological warfare agent and said molecularly imprinted polymer.