WO2009055702A1 - Time of death biosensor - Google Patents

Abstract

An apparatus and method for measuring the efficiency of a fumigation process. Also, methods and apparatus for measuring the respirational characteristics of a life form.

Description

TIME OF DEATH BIOSENSOR

CROSS REFERENCED TO RELATED APPLICATIONS

The application claims the benefit of priority to U.S. Provisional Patent Application Serial No. 60/982,170, filed October 24, 2007, all of which is incorporated herein by reference

FIELD OF THE INVENTION

Various embodiments of the present invention pertain to sensing systems that include a biological organism, and in particular, sensing systems that provide a signal corresponding to the respirational characteristics of the biological organism.

BACKGROUND OF THE INVENTION

For decades, structural fumigation has remained more of an art rather than a science and it has been performed without any type of direct efficacy measures.

Applicators typically overdose in order to ensure the success of a fumigation event. Methyl bromide has been the preferred fumigant in the structural pest control industry due to its fast reaction and high efficacy. In addition, the per pound cost of MeBr has been relatively low because it is a by-product of other bromide manufacturing processes. Therefore, overdosing has been regarded as an assurance measure for fumigation success rather than a misuse, and the importance of fumigation planning and monitoring has been largely overlooked.

Over the years, the fundamental aspects of the structural fumigation process have changed little. The main steps are: (1 ) the structure is sealed in order to make it as gas- tight as possible; (2) the fumigant is released and held in the structure for a certain period of time, called exposure time; and (3) when the desired exposure time has elapsed, the fumigated structure is aerated. Precision fumigation involves delivery of the precise amount of lethal concentration and exposure per unit volume at the target infestation spots with the minimum fumigant dosage rate. Precision fumigation techniques are aimed at optimizing fumigant use by maximizing efficacy and minimizing risk by integrating all factors affecting control, such as pest biology, temperature, exposure time, and improved sealing techniques. Precision fumigation requires preparation of a fumigation management plan, monitoring and controlling the fumigation progress according to this plan, and consideration of worker health and safety, food safety, insect resistance and environmental concerns.

Unlike MeBr fumigation, use of alternatives, such as ProFume^®, has been refined in order to optimize fumigant use. This typically includes intensive sealing of the structure, thorough planning of the fumigant introduction and monitoring sites, and utilization of state- of-the-art equipment for monitoring fumigant concentrations. For current fumigation practice, a fumigation event is considered successful when the lethal concentration (C) x time (t) product dosage has been reached. Lethal Ct products for different types and life stages of insect pests vary and are a function of temperature. Typically, eggs are more tolerant while adults are more susceptible. Therefore, the Ct product is a function of fumigant concentration, exposure time and insect mortality that is typically quantified based on fumigation experiments under laboratory conditions in which the fumigant concentration and fumigated space temperature are maintained constant . No mathematical/statistical model can ever perfectly describe all characteristics of a biological system. Therefore, when used in a practical situation, the Ct product carries a certain level of uncertainty with it and thus it is not a definite mortality indicator. Currently, insect bioassays are considered the ultimate proof of insect mortality, but they cannot be examined until the fumigation is completed. In the case of eggs, no simple method exists to determine vital signs. Thus, in order to confirm their mortality, bioassays have to be maintained post-fumigation under laboratory conditions and observed typically for up to two weeks in order to be confident that the egg stage was indeed killed.

MeBr depletes the stratospheric ozone layer and is classified as a Class I ozone- depleting substance. The amount of MeBr produced and imported into the United States is being incrementally reduced as part of a global phase-out effort based on the Montreal Protocol on Substances that Deplete the Ozone Layer . Based on the Montreal Protocol and the Clean Air Act, the United States was to reduce MeBr production and net imports incrementally from the 1991 baseline until a complete phase-out by 2005. Since 2005, MeBr continues to be available through the filing of critical use exemption (CUE) requests. Although viable alternatives such as ProFume^® (sulfuryl fluoride, SF), ECO2Fume^® (98% CO2 with 2% phosphine), a combination of heat treatment and ECO2Fume^®, and heat treatment alone have proven to be effective for the structural fumigation of flour mills, food manufacturing plants, and food warehouses, the total CUE amount for 2007 is still 6,230,655 kilograms. This represents 26.4% of the nation's 1991 baseline use. For 2008, the United States filed Critical Use Nominations (CUNs) at 25% of the 1991 baseline levels. Although this represents a continued reduction from earlier years, a large portion of agricultural soil and structural fumigation continues to rely on MeBr.

Various embodiments of the present invention provide novel and unobvious apparatus and methods for overcoming some of the problems and issues associated with fumigation. SUMMARY OF THE INVENTION

One aspect of the present invention pertains to an apparatus for indicating the respirational response of a life form. Further embodiments pertain to a chamber to contain the life form, the chamber including a breathable atmosphere. Yet other embodiments include a source of radiation, a transducer that emits radiation in response to receiving radiation from the source, the transducer being exposed to the atmosphere in the chamber; and a detector of radiation emitted by the transducer.

Yet another aspect of the present invention includes a method for detecting the viability of an respiring life form. Other embodiments pertain to providing a chamber to contain the life form within a respirable atmosphere, a source of radiation having an intensity that is modulated at a frequency, and a transducer having a characteristic that is altered in relation to the concentration of a respired gas in the chamber. Sill further embodiments include exposing the transducer to radiation from the source, emitting radiation from the transducer in response to exposing, and determining the respirational characteristics of the life form.

Another aspect of the present invention pertains to a system for fumigating a building with a gaseous biocide to kill a life form. The system includes a first plurality of detectors each providing a first signal corresponding to the local concentration of the biocide. Another embodiment of the present invention pertains to a second plurality of biosensors, each biosensor including a live organism, a chamber to contain the organism, an atmosphere within the chamber that is respired by the organism, and a sensing device that produces a second signal corresponding to the concentration of a respirational gas in the chamber atmosphere, and an electronic network hub receiving the first plurality of signals and the second plurality of signals.

It will be appreciated that the various apparatus and methods described in this summary section, as well as elsewhere in this application, can be expressed as a large number of different combinations and subcombinations. All such useful, novel, and inventive combinations and subcombinations are contemplated herein, it being recognized that the explicit expression of each of these myriad combinations is excessive and unnecessary.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of mitochondrial toxins on metabolic oxygen consumption measured in an individual fathead minnow egg (Pimephales promelas).

FIG. 2 shows the effect of mitochondrial toxins on metabolic oxygen consumption measured in an individual red flour beetle egg (Tribolium castaneum).

FIG. 3 shows an overview of a whole organism biosensor system according to one embodiment of the present invention.

FIG. 4 shows a basic calibration curve of the PtTFPP based oxygen optrode.

FIG. 5 shows data from experiments to improve the performance of PtTFPP as an optical oxygen sensor using frequency domain fluorescence lifetime.

FIG. 6 shows the IR absorption spectrum of Methyl Bromide.

FIG. 7 shows the schematic of an IRGA System according to one embodiment of the present invention. One end of the gas housing is mounted with a mid-IR Light Source (LED). FIG. 8 shows the combining of the RT-WOB and IRGA systems in a facility according to one embodiment of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the embodiments illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated device, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates. The use of an N-series prefix for an element number (NXX.XX) refers to an element that is the same as the non-prefixed element (XX.XX), except as shown and described thereafter. As an example, an element 1020.1 would be the same as element 20.1 , except for those different features of element 1020.1 shown and described. Further, common elements and common features of related elements are drawn in the same manner in different figures, and/or use the same symbology in different figures. As such, it is not necessary to describe the features of 1020.1 and 20.1 that are the same, since these common features are apparent to a person of ordinary skill in the related field of technology. Although various specific quantities (spatial dimensions, temperatures, pressures, times, force, resistance, current, voltage, concentrations, wavelengths, frequencies, etc.) may be stated herein, such specific quantities are presented as examples only, and are not to be construed as limiting.

Until now, no tool exists that enables pest control professionals to directly measure fumigation efficacy (i.e., actual percentage of insect mortality in the fumigated structure) while the structure is under gas. Although gas concentrations can be monitored, current fumigation monitoring approaches lack a definitive real-time indicator for fumigation success. One embodiment of the present invention pertains to a Fumigation Biomonitohng Network (FBN) 100 that includes real-time whole organism biosensor (RT-WOB) 20 technology. The RT-WOB 20 is based on the measurement of oxygen respiration, which is considered to be one indicator of life.

Mitochondrial oxygen consumption is one physiological signature of aerobically respiring organisms. Recently, the mitochondria has re-emerged in importance in biological research as the evidence supports that they are integral in regulating key processes in cell biology, including molecular metabolism, redox status, calcium signaling and programmed cell death in addition to energy production. These organelles can respond to intra- and extracellular cues independently and there is a highly coordinated communication system between organelle and nuclear signals that can greatly influence cell fate.

Monitoring system 100 includes one or more radiation or optical sensors (optrodes) instead of electrochemical microelectrodes to measure oxygen consumption. An optrode is the optical equivalent of an electrode that is constructed based on an analyte selective indicator molecule that changes its optical properties in response to analyte concentrations. Most optrodes that have been developed are based on fluorescence where excitation energy is provided to the indicator molecule which produces fluoresces (releases light of a specific wavelength) in proportion to analyte concentrations. Therefore, the transduction of the sensor output is in the form of photons, and the light produced by the fluorescence is detected by some method of photonic detection. In comparison to electrochemical sensors, the advantages of optrode technology includes: (1 ) O2 is not consumed by the sensor, thereby eliminating sensor artifacts; (2) there is no reference electrode; (3) the sensors are immune from external electromagnetic interference; and (4) they can be reduced down to nanometer sizes FIGS. 1 and 2 demonstrate operational testing of a 20 μm O2 optrode, based on PtTFPP immobilized in polystyrene.

FIG 1 shows the effect of mitochondrial toxins on metabolic oxygen consumption measured in an individual fathead minnow egg (Pimephales promelas). The developing embryo was approximately 1 day old at the time of the experiment and was 300 microns in diameter. Oxygen flux was non-invasively measured using a 20 micron self- referencing oxygen optrode at a frequency of 0.1 Hz, and over an excursion distance of 25 μm. At approximately 8 minutes after the recording was started the un-coupler CCCP was added to the bath and allowed to diffuse to the egg. The drug disrupts mitochondrial ATP production by allowing the chemiosmotic proton gradient to leak across the inner membrane. This causes the ETS to run full open, thereby causing metabolic oxygen flux to increase over the next 20 minutes. In this experiment it never reached a maximal value. The mode of action of rotenone, a common pesticide, is as a NADH dehydrogenase (complex I) inhibitor, and this directly inhibits the activity of the ETS and oxygen reduction.

FIG. 2 shows the effect of mitochondrial toxins on metabolic oxygen consumption measured in an individual red flour beetle egg (Tribolium castaneum). Oxygen flux was non-invasively measured using a 20 micron self-referencing oxygen optrode at a frequency of 0. 1 Hz, over an excursion distance of 25 μm. Baseline respiratory oxygen consumption was measured before application of biochemical inhibitors of mitochondrial cytochrome activity (Large panel = 1.0 mM KCN; inset panel = 1 mM DEA nitric oxide donor) were added to the bath solution (PBS pH 7.4). Blocking cytochrome c in the mitochondrial electron transport chain effectively blocks oxygen reduction. Cyanide partially blocks the mitochondrial ETS (large panel) as it is limited in penetrating the egg membranes, and insect metabolism is more resistant to cyanide toxicity. Nitric oxide (NO) is produced in solution by decomposition of the DEA donor. NO is a gaseous molecule and is highly permeable through the hydrophobic membranes of the egg, and therefore is more effective (inset) than CN in blocking oxygen consumption.

Six fumigation monitoring experiments were performed as part of regular fumigations of three commercial flour mills. The primary goals of these experiments were to gain insights into the fumigation process and acquire data for validating computer simulation models. In addition to fumigant concentrations, the environmental conditions both inside and outside of the fumigated facilities were monitored during the experiment at 15 - 20 locations throughout each mill. The efficacy of each fumigation was evaluated by insect bioassays of all life stages of red flour beetles (RFB) and Indian meal moth (IMM). The required dosage of a fumigant gas is influenced by the temperature at the site of the pest, the length of the exposure period, the rate at which the fumigant is lost from the structure (i.e., HLT), and the susceptibility and life stage of the pest to be controlled. While current fumigation practice assumes that these influencing parameters are uniform within the fumigated space and/or constant throughout the exposure period in order to determine the dosage rate (Ct product), our experimental results indicated that fumigation is a much more dynamic process. Table 1 summarizes the ranges of environmental conditions during each experiment. Effects of sealing on the environmental conditions in the fumigated structure were observed in all experiments. The inside temperature was always higher than the ambient temperatures and vice versa for the inside relative humidity. The very strong wind measured in the second experiment in Facility A occurred during a rainstorm that passed over the facility. Although the high velocity wind only lasted for approximately half an hour, it damaged sections of the external plastic sheeting seal of the mill, which led to a much lower HLT (6 hours) compared to the HLT (17 - 20 hours) from the first experiment. Facility C was fumigated with MeBr in the spring and sulfuryl fluoride (SF, or ProFume^® which is manufactured by DowAgroSciences, Indianapolis, Indiana, USA) in the fall. The HLT of the MeBr fumigation was estimated between 10 and 11 hours while that of the SF fumigation was 20 and 22 hours. However, the improved HLT was likely due to better sealing quality and lower wind speeds. The average wind speeds during the MeBr and SF fumigations were 3.4 m/s and 2.6 m/s, respectively. Facility B was the least air-tight structure. In spite of intensive sealing and relatively mild wind, the HLTs for both fumigations in this facility were only 5 to 6 hours. These results showed variability in the fumigation-related parameters that had impact on the success and effectiveness of each fumigation.

Table 1. Summary of environmental conditions during fumigation experiments performed in three flour mill facilities.

Despite the dynamics of the fumigation process, no data has been found in the literature or used by industry that describes the relationship between insect mortality and dosage application rates under varying conditions. According to the Fumiguide™ software, the Ct product of ProFume^® required to kill RFB increases from 594 oz-hr/Mcf at 86⁰F to 1455 oz-hr/Mcf at 68⁰F, given that HLT = 12 hours and the exposure time = 24 hours. Temperature inside a sealed structure is not uniform and could potentially decrease by up to 5O⁰F. For example, during Experiment #5 the temperature inside the fumigated structure deceased from a range of 82-97⁰F to 68-73⁰F while the outside temperature was between 43-5O⁰F. This implies that this particular fumigation could have been a failure if the fumigation had been stopped when the achieved Ct product reached the required Ct product determined based on the high temperature range. On the other hand, overdosing the fumigant based on the Ct product at the low temperature range would result in excess gas emission and use. Table 2 compares the theoretical and actual values of the Ct product and total fumigant usage as well as the insect mortality of all six experiments. The theoretical Ct product and total fumigant use were recommended by the Fumiguide™ software to kill RFB eggs, which are the most tolerant species, based on the temperature ranges in Table 1. The mortality of all other life stages was 100% and thus the results are not shown. The RFB egg mortality was classified into two types. Type I mortality included only unhatched eggs. Type Il mortality included unhatched eggs and eggs that hatched but failed to reach adulthood. Although in most cases (Experiments #1 , 3, 4 and 6) the recommended Ct products were achieved, Type I mortality indicated that some eggs did not die during the fumigation. However, most of these eggs hatched and died before they developed to adulthood (i.e., 99-100% Type Il mortality).

In two fumigations 11 -14% of eggs survived and developed into adulthood. The mechanism of this delayed mortality has not been understood. While the achieved Ct product did not reach the recommended value in Experiment #2, in Experiment #6 the achieved Ct product fell within the recommended range. However, the Type Il egg mortalities for both cases were approximately the same. This emphasizes uncertainty in determining insect mortality base on the Ct product approach. In Experiment #1 , although 100% kill (Type II) was achieved, the fumigant was overdosed by 25%.

Table 2. Theoretical and actual values of the Ct product, total fumigant use, and insect mortality of all fumigation experiments.

Recommended Achieved Recommended Released

1 589¹ 687 - 1127 2401¹ 3000 41 100

2 628¹ 250 - 510 4854¹ 2375 N/A 89

3 644¹ 668 - 907 1365¹ N/A 91 99

4 653 - 758 790 - 990 1396 - 1620 1500 94 99

5 N/A 150 - 310 N/A 695 N/A 100

6 595 - 1193 800 - 1150 359 - 719 875 74 86

¹The Fumiguide ^{I M} software gives the same values for the entire temperature range.

A Real-Time Whole-Organism Biosensor (RT-WOB) 20 according to one embodiment of the present invention, as shown in FIG. 3, is based on the application of optical oxygen sensors (FIG. 4) to monitor the respirator oxygen consumption of individual insects. Although what is shown and described herein is a biosensor for sensing the response of an insect, other embodiments of the present invention contemplate biosensors that include life forms other than insects. Further, although the biosensors shown and described include one or more eggs in a chamber, other embodiments of the present invention contemplate the use of insects or other life forms at other stages in the life cycle. Further yet, some embodiments of the present invention contemplate the use of a plurality of chambers, in which some chambers include a life form of a species at one stage in the life cycle, and other chambers include life forms of the same species at a different stage in the life cycle.

Optical sensors are different than electrochemical methods and in some embodiments present reduced incidence of fouling, less noise and less drift. Optical oxygen sensors also do not consume O2 during measurement as compared to electrochemical sensors. Using optical methods it is possible to separate the passive fluorescent sensor from the instrumentation hardware. Although what has been shown and described is the use of optical oxygen sensors, the present invention also contemplates those embodiments in which the measurement of any respirational gas is performed by any method that does not consume the gas. Although the term "optical" is used, various other embodiments pertain to measurement of any wavelength of electromagnetic radiation.

FIG. 3 shows an overview of a whole organism biosensor system 20 according to one embodiment of the present invention. The optical oxygen sensors in one embodiment are based on immobilization of platinum tetrakis (pentafluorophenyl) porphyrin (PtTFPP) as the oxygen indicator dye, immobilized within a polystyrene membrane substrate. However, other embodiments of the present invention pertain to optical oxygen sensors using other types of indicator dyes, and other substrate materials. The sensor membrane 28 is incorporated into a wall of the plastic chamber 24, containing individual microchambers for each life form. Further yet, the sensor module can be immobilized within a polystyrene membrane in the bottom of micro chambers within a plastic cartridge system. Although what is shown and described is a wall of the chamber that includes an indicator dye, yet other embodiments of the present invention contemplate biosensors in which the apparatus measuring the respirational gas is a separate component within the chamber, or is a separate component molded or otherwise placed within a wall of the chamber. Each chamber 24 is in fluid communication with the ambient atmosphere of the room of the building. Room ambient air (which will include the fumigant) is provided at a relatively low flowrate to each chamber 24. The rate at which room ambient air A enters the respirational atmosphere of a particular chamber 25 can be by natural convection currents within the room, the forced ventilation provided to the room (i.e., such as through the HVAC ducts), or by a nearby fan. The sensor membrane measures oxygen concentration levels associated with each insect 22 in the cartridge. As the life form 22 within the chamber 24 respires from chamber atmosphere 25, the concentration of a respirational gas (such as oxygen or carbon dioxide) is altered within the chamber, and therefore alters the response of transducer 28.

The sensor itself will be monitored by hardware including a blue LED to provide fluorescent excitation to the PtTFPP, a longpass filter 29 for fluorescent emission at 650 nm, and a radiation detector 30 to transduce the fluorescence signal into metabolic signals. Many components for this system are available as off the shelf devices and can be integrated through fabrication of custom instrument PCBs .

FIG. 4 shows a basic calibration curve of the PtTFPP based oxygen optrode. The fluorescent properties of the dye change in response to collisions with oxygen molecules. This can be measured in terms of fluorescent quenching (intensity) or as a change in the fluorescent lifetime of the dye. Fluorescent lifetime provides a more reliable indicator as it is not subject to sensor calibration drift because of photobleaching of the dye. Fluorescent lifetime (as phase angle) is measured here using the frequency domain lifetime approach using a commercial lock-in amplifier. Calibration solutions were made using deionizer water bubbled with known gas mixtures. The chamber 24 is adapted and configured to closely contain the life form 22. If the interior volume of chamber 24 is too large in relationship to the respirational rate of the life form and the size of the life form, then the change in the concentration of respirational gases in the atmosphere of the chamber will be mixed with too large a quantity of non-respired atmosphere. Therefore, the concentration of the respired gas would be too low, and thus the alteration in fluorescence characteristics of the transducer may be too small to either promptly and/or accurately measure. Therefore, the volume of chamber 24 is preferably sized in accordance with the size of the life form, and the respirational characteristics (especially the respirational flowrate) of the life form, and the desired response rate of the biosensor 20. Preferably , the ratio of the volume of the chamber to the volume of the life form is less than about 100:1 . In some embodiments the volume of the chamber to the volume of the life form is less than about 30:1 and in yet other embodiments the ratio is less than about 10:1.

An optical sensing system 20 includes: (a) a radiation or light source 26, (b) a transducer element 28 converting radiation into a detectable signal (Fluorophore), and (c) a detector unit 30 detecting and converting the change in optical properties of the sensor into a read-out. There is wide variety of light sources 26 available, for example lasers and broadband-emitting light sources like halogen or xenon lamps. LEDs as light sources offer the advantages of small size, low power consumption and negligible heat production. The LED light source 26 is preferably inexpensive. One advantage of LEDs is their narrow emission spectra which can now be purchased to match the excitation spectra of the fluorophore. In one embodiment, system 20 uses commercially available 505 nm blue LEDs that match the fluorescent excitation for the platinum tetrakis (pentafluorophenyl) porphyrin (PtTFPP) oxygen indicator dye.

A modulated blue LED 26 illuminates chamber 24 containing egg 22 and O2 sensitive membrane. The fluorescent O2 indicator 28 emits red light fluorescence that has a lifetime that is proportional to the O2 concentration in the egg chamber 24. A long pass filter 29 lets modulated red fluorescence pass to detector 30 to transduce the signal related to the oxygen/egg viability in the chamber. Although what has been shown and described is a biosensor 20 in which a blue source causes red fluorescence of an indicator of a respirational gas, the present invention is not so constrained, and contemplates any system in which radiation of a first wavelength is used to induce fluorescence at a second, different wavelength. Further, although what has been shown and described is a sensor in which the fluorescence lifetime is proportional to the concentration of the respirational gas, other embodiments contemplate a fluorescing sensor in which the magnitude of fluorescence corresponds to the concentration of a respirational gas in the chamber.

There is also a large variety of optical detection units 30 available. For very small optical sensors (μm-nm size) a Photo-Multiplier Tube (PMT) can be used. While Charge Coupled Devices (CCDs) are less sensitive than a PMT, they are an inexpensive alternative depending on the application. System 20 includes the individual oxygen sensors that are in the mm size range, and in some embodiments uses a CCD based system. Today CCD based cameras are even integrated into cellular phones, and therefore are small in size and commercially available at low prices. Further, each pixel of the CCD 30 can be utilized as a sensor . Pixels on the CCD can be grouped together to collect optical data from a single sensor spot within the cartridge, correlating with an individual insect. The CCD can be reconfigured dynamically in software to match different cartridge configurations that could be developed for different insect biosensors. This provides more freedom in terms of spatial data acquisition and statistical sampling for analyzing the data. The CCD based detection system is more favorable for high- throughput systems such as the RT-WOB.

The RT-WOB 20 according to one embodiment of the present invention includes a polydimethylsiloxane (PDMS) cartridge 21 with a plurality of sample chambers 24. In some embodiments, cartridge 21 having multiple chambers 24 is easily separable from a housing that contains the radiation sources 26, filters 29, and radiation detectors 30. Cartridge 21 can be separately fabricated and populated with eggs, and then brought to the site of fumigation to be inserted into a housing adapted and configured to accept the cartridge and separately irradiate and detect the individual responses of the life form 22. PDMS is an elastomer which is available commercially. The size of the cartridge 21 and the sample chambers 24 can be designed and controlled by direct fabrication, such as by casting the un-polymehzed material into a machined mold. The individual insect sample chambers 24 in the cartridge will be modified by the application of a polymer- indicator matrix that contains the oxygen indicator dye, platinum tetrakis (pentafluorophenyl) porphyrin (PtTFPP). PtTFPP has two excitation peaks (505 and 541 nm) and an emission peak at 650 nm.

The polymer-indicator matrix includes a solvent, a polymer matrix, and the PtTFPP. The solvent dissolves the polystyrene polymer matrix with the PtTFPP dye. Once the solvent evaporates the polystyrene provides an oxygen permeable solid matrix for the PtTFPP as it is immobilized in the bottom surface of the sample chamber 24. Yet other embodiments of polymer-indicator mix provide the optimum optical signal to measure the fluorescence lifetime of the PtTFPP by using titanium (II) oxide micro and nanoparticles (FIG. 5). FIG. 5 shows data from experiments to improve the performance of PtTFPP as an optical oxygen sensor using frequency domain fluorescence lifetime. The PtTFPP dye (10 mg) was immobilized in polystyrene using Chloroform as a solvent. To this base membrane solution different amounts of TiO2 particles (1 -2 microns) were added to change the optical scattering within the final polymerized membrane. According to preliminary data, it appears that the 1 -2 μm sized titanium (II) oxide particles improve sensor performance. Adding Titanium (II) Oxide particle to the polymer-indicator mix improves its sensitivity and signal to noise ratios as indicated by an increase in signal amplitude.

Fluorescence quenching refers to any process decreasing the luminescence intensity of a probe (signal transducer), including collisional quenching or energy transfer. Oxygen is known to act as a quencher of luminescence of many fluorophors . Thus, one class of oxygen sensors is based on the decrease of the fluorescence signal (intensity) of an oxygen-sensitive material, i.e., the indicator dye, as a function of oxygen concentration . Several oxygen quenchable fluorophores, which are suitable as indicators in optical sensors, are reported in the literature, mostly polycyclic aromatic hydrocarbons and transition metal complexes. Different embodiments of the present invention contemplate the use of any oxygen quenchable fluorophor. Further, yet other embodiments of the present invention contemplate the use of transducers that, instead of having fluorescence quenched by oxygen, provide fluorescence that is intensified by the presence of oxygen. Further yet, other embodiments of the present invention contemplate radiation sensors whose fluorescence characteristics are either quenched or intensified by the presence of other respirational gases, such as carbon dioxide. Preferably, the transducer does not consume or alter the respirational gas during the radiation transduction process.

Usually, the fluorescence quenching is described by the Stern-Volmer equation:

- = i + k_qτ₀[Q] (1 )

where τ₀ is the lifetime of non-quenched fluorophore, τ is lifetime of the fluorophore in the presence of the quencher, k_q is the bimolecular quenching constant and [Q] is the concentration of quencher Q.

While optical dye fluorescent intensity can be measured for optical sensing, this is subject to calibration drift due to photobleaching of the dye during use. Instead fluorescence lifetime can be used and is not subject to photobleaching artifacts. The fluorescence decay time τ₀ of a fluorophore is defined by the average time the molecule spends in the excited state prior to return to the ground state. Two methods are used for the determination of luminescence decay times, namely time-domain and frequency- domain lifetime measurements. In some embodiments of the present invention, frequency domain is the used because it can be less expensive to implement when compared to time domain methods. In the frequency domain method the sample is excited by sinusoidally modulated light and the lifetime of the fluorophore causes a time lag between absorbance and emission (phase modulation). This is expressed by the phase shift θ (FIG. 4) between excitation and emission phases, and the decreased emission intensity relative to the incident light, called demodulation . The luminescence decay time τof the luminescent probe in atmospheres of defined oxygen concentration can be calculated from the equation;

where θ is the phase-shift between excitation and emission, τ is the lifetime of the fluorophore in the presence of the quencher and /_mod is the modulation frequency of the illuminating light source (LED). The time period of the modulation signal is preferably higher than the fluorescence lifetime of the fluorophore. Based on the published lifetime of 59 μs for PtTFPP, /_mod is set to 5 kHz to achieve a time period of 200 μs, which is slow enough for substantially complete fluorescence of PtTFPP.

When IR light strikes a substance, the radiation is transmitted, reflected or absorbed in varying degrees, depending upon the substance and the wavelength of radiation. A molecule can only absorb energy from a photon if the energy matches precisely the "energy state" of the molecule. Inert gases (He, Ne, Ar, Kr, Xe, Rn) or diatomic molecules composed of like atoms (H₂, O₂, Cl₂, and N₂) only oscillate in the high energy state. They are transparent to IR Radiation. More complicated molecules like CO₂, CH₄, CH₃Br (MeBr) and SO₂F₂ absorb radiation in the IR region. Each gas exhibits a very specific set of wavelengths, and this provides the selectivity to measure a particular gas molecule which can be easily controlled by the wavelength of radiation. Various IR detection schemes for different gases have been reported previously. The IR absorption spectrum for MeBr (FIG. 6) shows characteristic absorption peaks at 3.3, 7, 8 and 11.5 μm in the IR spectrum. The plotted wavenumber is the reciprocal of wavelength.

The IRGA System 60 (FIG. 7) is based on Infra-Red (IR) detection of gas concentration governed by the Beer-Lambert Law. The main advantage of IR based gas sensing is that the detector does not interact directly with the sample and has very high specificity. The major functional components of this system will be simplified for specific analyte detection (MeBr). Commercially available similar systems are expensive (thousands of dollars) due to their complicated design and expensive components needed to facilitate broad detection of multiple gases. However, due to advancement in semiconductor process technology, a dedicated infra-red gas analyzer (IRGA) system 60 for specific analytes, for example MeBr, can be fabricated which are not only less complex in terms of hardware (miniaturization) but also much more cost-effective (hundreds of dollars).

FIG. 7 shows the schematic of the dedicated IRGA System 60 according to one embodiment of the present invention. One end of the gas path 66 includes a mid-IR Light Source (LED). The other end contains the detector (Photodiode). Ambient gas A within the room being fumigated flows over and within the RT-WOB cartridge, and is fed into the chamber using a pump. This fumigated gas interacts with the IR radiation of the source. Upon interaction with the radiation, the gas molecules absorb a certain amount of radiation which is proportional to the fumigant gas concentration. The unabsorbed radiation is detected by the Photodiode. The absorbed radiation is proportional to the concentration of gas present in the chamber. Although what is shown and described is an infrared method of detecting a fumigant gas, the present invention contemplates any sensor that remotely provides a signal corresponding to the concentration of the fumigant gas.

Components of an IRGA System 60 can include: (a) an IR source 62, (b) a detector 64 (such as a Photodiode), (c) the gas path 66 includes an inlet 69 and an outlet 70 between IR source and detector for the gas molecules to interact with the radiation, and (d) the optical elements such as filters 68. There are a variety of IR sources 62 available including IR LEDs. IR LEDs capable of emitting up to 7 urn IR radiation are available commercially due to advancement in the semiconductor material and manufacturing. Similarly, photodiodes capable of operating and providing high performance in the mid-IR range are also available, and can be purchased to match the emission of the IR LEDs. Additionally, IR LEDs can be modulated electronically, therefore reducing the need of using choppers for measurement of the reference signal. The LED emission wavelength substantially matches the absorption wavelength of MeBr and the wavelength of photodiode where maximum transmittance can occur. As shown in FIG. 6, the 7-8 μm wavelength regions are not easily resolvable.

Additionally, the photodiodes for this wavelength range are usually cooled for better performance. However, IR LED sources and photodiodes are easily available commercially to operate in the 3.3 μm wavelength and do not have to be cooled. Therefore, an IRGA System 60 according to one embodiment of the present invention for MeBr operates at a wavelength of 3.3 μm. These components are inexpensive and with additional circuitry to control the optoelectronic components, a dedicated and inexpensive MeBr sensor is provided. Using similar methodology, other IRGA Systems for MeBr alternatives such as sulfuryl fluoride (SO2F2) are also contemplated . However from the literature, the IR absorption spectrum of SO2F2 is in the 11 - 13 μm wavelength range and currently, IR LEDs for this wavelength region do not yet exist. Based on the developments in semiconductor manufacturing and LED technology it is reasonable to expect that these components will be available. For example, the 3.3 μm wavelength IR LEDs which are proposed for the development of a dedicated miniature MeBr IRGA were not available just a few years ago.

The Fumigation Biomonitoring Network 100 includes one or more IRGA systems 60, one or more biosensors 20, a digital signal processor, and a receiving station. Each gas analyzer 60 is located proximate to a biosensor 20 and together comprise a measurement node of system 100. Referring to FIG. 8, a plurality of pairs of gas analyzers 60 and biosensors 20 are shown providing electronic signals to respective digital signal processors 90. Processors 90 perform one or more of the processing algorithms described herein. The output of processors 90 is provided to a wireless transmitter 92. Although what has been shown and described is a distributive processing system, the present invention also contemplates those embodiments in which signals from gas analyzers 60 and biosensors 20 are provided to a common digital signal processor.

Data from the transmitters 92 is provided, preferably by a wireless system 93, to a receiving station 94. An operator receives and interprets the data provided by the analyzer 60 and biosensors 20, and determines the status of the fumigation process.

In one embodiment the biosensor 20 each hold multiple target insect samples 22 and chambers 24 (8-32 depending on size of the insect) and are deployable individually in the field. Using red flour beetle eggs, one embodiment of system 100 can accommodate 32 samples in each individual FBN node device, and can be placed at strategic locations and monitored while the mill is fumigated. The real-time data is relayed back to the wireless network hub 94 where the fumigant concentrations and mortality of the sample insects can be observed by the researchers, facility owners and fumigators. This real-time mortality analysis preferably includes an algorithm for assessing the status of the fumigation process and in some embodiments also includes one or more computer simulation models of the fumigation process.

Commonly used signal conditioning techniques that are applied to raw sensor data include signal amplification and filtering . Signal amplification uses electronic components to increase the signal so that the noise and actual data can be differentiated. Filtering is the removal of unwanted components of a signal such as electromagnetic noise and enhancement of the desired signal of interest. After signal amplification and filtering, the signal, the analog signal is subject to analog-to-digital conversion (ADC) using discrete electronic components. This processed signal can then be stored, or transmitted over a wireless network. Signal conditioning and processing from analog to digital is known as digital signal processing (DSP).

For the RT-WOB frequency domain fluorescence lifetimes are measured using a processor 90 including a lock-in-amplifier (LIA) using phase-sensitive detection (PSD). To facilitate PSD the LIA applies amplification and filtration preferably at a specific modulation frequency, with other frequencies are filtered out . In the frequency domain lifetime method a reference signal is used to excite the light source. The emission signal collected by the detector is sent to the LIA and the phase-shift is measured. This phase- shift is then correlated with the concentration quencher. LIA functionality is programmed onto a field programmable gate array (FPGA). FPGA is a semiconductor device that contains programmable logic components which can be programmed to perform desired functionality such as signal amplification, filtering and PSD. FPGAs are commercially available with software support development kits provided. FPGAs are reusable, and reprogrammable within a footprint that reduces to a typical printed circuit board. Since most signal conditioning is done via software, noise is reduced by minimizing the electronic components.

WSNs have advanced in the last decade, and are used in precision agriculture where spatial data is acquired. Applications in agriculture now allow data such as temperature, humidity, pressure, sunlight and soil data to be recorded over a wide geographic area and relayed back to a central hub where the data is recorded and analyzed. For real-time support, it is desirable to transmit experimental data over a WSN. Although a wide variety of protocols and architectures exist, the system level concept of WSN (as shown in FIG. 8) follows the general format of: 1 ) multiple sensors (20, 60) are installed over the area of interest; 2) recorded sensor data is processed and sent to a gateway; and 3) the gateway relays sensor data to a central hub. The central hub is a hand-held personal computer from which the data can be analyzed.

In one embodiment, real-time data transmission from the RT-WOB 20 and IRGA 60 sensors are supported with the Crossbow Wireless Sensor Networks. FIG. 8 shows the overall integration at the component level. FIG. 8 shows the raw signals from the RT-WOB and IRGA systems across the facility are fed into the Customized DSP FPGA LIA. It performs signal conditioning and processing before sending it to the Crossbow wireless module (MICAz). The MICAz operates at the ISM band frequency of 2.4 GHz and transmits data to the Cross Wireless Gateway (MIB520). MIB 520 is a USB Gateway that is connected to the PC where data will be downloaded, analyzed and used for determination of efficacious fumigant concentration.

The operation of the sensors and system described herein will now be described with reference to the fumigation of a flower mill. In addition to describing operation of various embodiments of the inventions, reference will also be made to the additional apparatus and methods which would be useful in verifying the on-site performance of these embodiments. The RT-WOB sensors 20 performance can be assessed against traditional bioassays. The IRGA sensors' performance can be assessed against a calibrated fumiscope. In the case of flour mills, insect population in each structure can be monitored for three months pre- and post-fumigation using insect traps. The pre- and post-fumigation insect population data can be used to assess the pest infestation buildup before the fumigation and the rebound after the fumigation.

Fumigation is preferably conducted using the standard fumigation protocol based on the fumigant's product label. The structure is cleaned and sealed with fumigation tape and plastic sheeting. Fumigant introduction lines and fans are placed at locations at which fumigant distribution time and usage are optimized. IRGA sensors 60 and temperature sensors are placed at locations including where high/low gas concentrations may occur. Fumigant monitoring lines are preferably placed proximate to the locations of the IRGA sensors 60 and run to an automated multiplexing valve hub connected to the fumiscope and placed outside the facility . The RT-WOBs and the bioassays are placed at the same locations as the gas and temperature sensors as well as other locations at which insects are more likely to survive. Such locations may be the inside of milling equipment (e.g., sifters), in the proximity of leaky areas, or under flour dust residue. In order to properly resemble insect distribution in the structure, four RT- WOB sensors 20 are placed in each floor of the structure. For worker safety purposes, the site surrounding the structure and any work area near the structure will be continuously monitored by low-range gas detectors to ensure that concentration levels do not exceed the safe limit (1 ppm) specified in the label.

After the fumigant is introduced into the room ambient air A, the status of all sensors (concentration, temperature, and insect mortality) are continuously recorded throughout the fumigation and later analyzed for evaluating the accuracy of IRGA sensors versus fumiscope readings and efficacy of the fumigation. Real-time data from sensors 20 and 60 is used as feedback to regulate fumigant dosage. The target dosage (Ct product) is based on the fumigant's product label. The actual achieved Ct product at each monitoring location is continuously computed. The actual achieved Ct value at which the RT-WOB sensors indicate insect mortality is documented and compared to the target Ct value. Once a sufficient member of RT-WOB sensors indicate insect mortality, the fumigation is considered successful and the structure is aerated until the gas concentrations in the structure are below the safe limit (1 ppm).

While the inventions have been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected.

Claims

WHAT IS CLAIMED IS:

1. An apparatus for indicating the response of an oxygen-breathing life form to a biocide, comprising: a chamber to contain the life form, said chamber including a breathable atmosphere, the life form being exposed to the biocide within said chamber; a source of radiation in a first spectrum; a transducer that emits radiation in a second spectrum in response to receiving radiation in the first spectrum from said source, the second spectrum being different than the first spectrum, said transducer being exposed to the atmosphere in said chamber; and a detector of radiation emitted by said transducer; wherein the emission of radiation by said transducer varies in response to the presence of oxygen within said chamber.

2. The apparatus of claim 1 wherein the transducer is included in a portion of a wall of the chamber.

3. The apparatus of claim 1 wherein the life form is an insect.

4. The apparatus of claim 3 wherein said chamber is adapted and configured to closely contain the insect.

5. The apparatus of claim 1 which further comprises a filter for obstructing a predetermined range of wavelengths, said detector receiving radiation from said filter.

6. The apparatus of claim 1 wherein the transducer is an optrode.

7. The apparatus of claim 1 wherein said chamber has at least one wall and the wall includes an oxygen indicator dye.

8. The apparatus of claim 7 wherein the dye is PtTFPP.

9. The apparatus of claim 1 wherein said transducer does not consume oxygen.

10. The apparatus of claim 1 wherein said source is pulse width modulated at a frequency.

11. The apparatus of claim 10 which further comprises a processor for measuring a phase angle between the source radiation and the emitted radiation at the frequency.

12. A method for detecting the viability of a respiring life form, comprising: providing a chamber to contain the life form within a respirable atmosphere, a source of radiation having an intensity that is modulated at a frequency, and a transducer having a fluorescent characteristic that is altered in relation to the concentration of a respired gas in the chamber; exposing the transducer to radiation from the source; fluorescing radiation from the transducer in response to said exposing; comparing the source radiation to the fluoresced radiation; and determining the respirational characteristics of the life form by said comparing.

13. The method of claim 12 wherein said source is modulated at a frequency less than about 1 Hz, and said comparing is by measuring a phase difference between radiation from the source and radiation from the transducer.

14. The method of claim 12 wherein the respired gas is oxygen.

15. The method of claim 12 wherein the alteration in the fluorescent characteristic is a quenching of fluorescence.

16. The method of claim 15 wherein the quenching is proportional to the concentration of the respired gas.

17. The method of claim 12 which further comprises filtering the emitted radiation before said comparing.

18. The method of claim 12 wherein the life form is an insect and the chamber is adapted and configured to closely contain the insect.

19. A system for fumigating a building with a gaseous biocide to kill a life form, comprising: a source of biocide that is distributed within the building ; a first plurality of detectors each one providing a first signal corresponding to the local concentration of the biocide, each one of said first plurality of detectors being located in different locations within the building; a second plurality of biosensors, each one of said second plurality of biosensors being located in different locations within the building, each said biosensor including a live organism representative of the life form, a chamber to contain the organism, an atmosphere within the chamber that is respired by the organism, and a sensing device that produces a second signal corresponding to the concentration of a respirational gas in the chamber atmosphere without consuming the respirational gas; and an electronic network hub receiving said first plurality of signals and said second plurality of signals.

20. The system of claim 19 wherein the life form is a species of insect and the organism is an egg of the insect.

21. The system of claim 19 wherein the ambient atmosphere within the building flows into each chamber and flows past each detector.

22. The system of claim 19 wherein the respirational gas is oxygen.

23. The system of claim 19 wherein a first portion of said biosensors each include a first organism, a second portion of said biosensors each include a second organism, and the first organism is different than the second organism.

24. The system of claim 19 wherein the first organism and the second organism are from the same species and the first organism is at a different stage of the life cycle than the second organism.

25. The system of claim 19 wherein the sensing device is an optrode and the respirational gas is oxygen.

26. The system of claim 19 wherein the first signal corresponds to the infra- red response of the biocide.