Abstract:
A measurement system for use with fluorescent chemosensors has multiple stimulus light sources each coupled to at least one sensor. Multiple sensors each receiving light from a different light source connect to each of one or more photodetectors. A processing device drives the light sources in a time-division multiplexed manner, and reads the photodetector at an appropriate time for each sensor. The processing device calibrates the sensor readings and provides them in a way that is identified to the associated sensor.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application claims priority from U.S. Provisional Patent Application No. 61/159,361, filed 11 Mar. 2009, which is incorporated herein by reference. 
     
    
     GOVERNMENT RIGHTS CLAUSE 
       [0002]    This invention was made with government support under Grant No. BES0529048 awarded by the National Science Foundation. The government has certain rights in the invention. 
     
    
     BACKGROUND 
       [0003]    There are many new chemo-optical sensing devices that are read optically. 
         [0004]    Many of these sensors involve a chemosensor component that is photometrically interrogated by an electro-optical component. In these sensors, the electro-optical component may measure optical changes at the chemosensor component such as absorption changes at ultraviolet and/or visible wavelengths (e.g. color changes), fluorescent and/or phosphorescent emissions, and optical scattering properties. When fluorescent reagents are utilized a fluorescent substance is excited by stimulus light at a stimulus wavelength, and one or more substances in the chemosensor component absorbs this stimulus light and emits light of a longer wavelength. 
         [0005]    Such sensors may have enzymatic components in their chemosensor component, such as the enzymatic biosensors described in U.S. patent application Ser. No. 12/358,140, hereinafter the &#39;140 application, filed 22 Jan., 2009, and entitled ENZYMATIC BIOSENSORS WITH ENHANCED ACTIVITY RETENTION FOR DETECTION OF ORGANIC COMPOUNDS, which is hereby incorporated herein by reference. 
         [0006]    For example, photoluminescence (PL), a generic term for both fluorescence and phosphorescence, may be employed for sensing by exciting a sample and directly looking for the PL spectrum of the analyte or by indirectly observing changes in the PL of another species affected by the analyte. Optical enzymatic biosensors typically use an indirect mechanism whereby the products of the reaction modify the PL efficiency of nearby dye molecules. For example, conversion of toluene by a monooxygenase consumes dissolved oxygen in the proximity of an oxygen-sensitive ruthenium-based dye thereby altering the dye&#39;s PL efficiency and lifetime. Higher analyte levels result in higher reaction rates and thus depleted oxygen levels, reducing oxygen alters phosphorescent emission of the ruthenium dye under constant excitation power. 
         [0007]    Each chemosensor or biosensor typically has an optode for coupling light to and from optical fibers to a sensor component. Chemosensors of particular interest herein are biosensors in that they incorporate a biological component in the sensor component. The biological component may be prepared of living organisms embedded in other materials, or may be made of isolated enzymes and/or antibodies combined with other materials. 
         [0008]    Many prior sensors have a one-to-one relationship between the electro-optical component and the chemosensor component. These prior sensors typically have an interrogation light source coupled directly or through an optical fiber to the chemosensor component, and an electro-optical detector component coupled directly or through an optical fiber to the chemosensor component. 
         [0009]    For example, consider the prior-art sensing device  100  illustrated in  FIG. 1 ; this device has a chemosensor element  102 , such as those known in the art or described in the &#39;140 application that undergoes a change in fluorescent properties with analyte concentrations in its environment. A stimulus light source  104  provides light at a stimulus wavelength suitable for stimulating fluorescence in chemosensor element  102 . Stimulus light source  104  may be a laser, may be a light-emitting diode, or may be another light source as known in the art. Light from light source  104  passes to chemosensor element  102 , and stimulates fluorescent light emissions at a fluorescence wavelength that is typically longer than the stimulus wavelength. In embodiments where light source  104  emits significant light at the fluorescence wavelength, a wavelength-selective device  106 , such as a high-pass optical filter, is interposed between light source  104  and chemosensor element  102  to block light at the fluorescence wavelength. 
         [0010]    In this device, fluorescent light emitted by chemosensor element  102  passes through a second wavelength-selective device  108 , typically a filter, that blocks light at the stimulus wavelength while passing light at the fluorescence wavelength. Light passed by wavelength-selective device  108  enters a photodetector  110 . A processing device  112  uses photodetector  110  to make readings of light at the fluorescent wavelength, applies any necessary correction factors, and provides readings of analyte concentrations. 
         [0011]    Many chemosensor elements  102  known in the art provide faint fluorescent light at some analyte levels of interest, in part because analyte levels of interest may be quite low. For example, it is desirable to detect substances such as the highly toxic organophosphate Sarin at levels that are below those that cause harm to most mammals. In order to accurately measure such faint fluorescent light, sensitive photodetectors  110  may be required, including such photodetectors as avalanche photodiodes and photomultiplier tubes. Such sensitive photodetectors may be rather costly. 
       SUMMARY 
       [0012]    A measurement system for use with fluorescent chemosensors has multiple stimulus light sources each coupled to at least one sensor. Multiple sensors each receiving light from a different light source connect to each of one or more photodetectors. A processing device provides for driving the light sources in a time-division multiplexed manner, and reads the photodetector at an appropriate time for each sensor. The processing device calibrates the sensor readings and provides them in a way that is identified to the associated sensor. 
         [0013]    A measurement system has stimulus light sources and sensors coupled in pairs by a stimulus optical fiber. Each sensor has an optode coupled to a photoluminescent chemosensor component for emitting fluorescent light at a fluorescent wavelength when illuminated by light at the stimulus wavelength. The sensor provides emitted fluorescent light dependent upon an analyte concentration. Multiple chemosensors are coupled to a common photodetector to measure the emitted fluorescent light by sensing optical fibers. A portion up to all of the stimulus and sensing optical fibers may be combined in a single fiber connected to a stimulus light source and photodetector by a 2×2 fiber coupler, bifurcated fiber assembly, dichroic filter, or other optical device for combining two optical paths that may be at different wavelengths. A processing device is provided for analyzing the emitted fluorescent light and controlling the light sources. 
         [0014]    A method of monitoring a level of an analyte has a cycle of providing light to a first sensor from a first stimulus light source at a stimulus wavelength; measuring light at a fluorescent wavelength from the first sensor with a photodetector; turning off the first stimulus light source; providing light to a second sensor from a second stimulus light source at the stimulus wavelength; and measuring light at the fluorescent wavelength from the second sensor with the photodetector. In this method, each sensor has an optode coupled to a photoluminescent chemosensor component for emitting fluorescent light at a fluorescent wavelength when illuminated by light at the stimulus wavelength, the emitted fluorescent light being dependent upon an analyte concentration. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0015]      FIG. 1  is a block diagram of a prior art sensing device. 
           [0016]      FIG. 2  is a block diagram of an embodiment of a multiplexed system having multiple sensing devices. 
           [0017]      FIG. 3  is a timing diagram of operation of the system of  FIG. 2 . 
           [0018]      FIG. 4  illustrates decay with time of fluorescence from a sensor. 
           [0019]      FIG. 5  illustrates measurements of decay time of fluorescence for determining a sensor reading. 
           [0020]      FIG. 6  is a block diagram illustrating an embodiment of an expanded multiplexed system having multiple sensor groups. 
       
    
    
     DETAILED DESCRIPTION OF THE EMBODIMENTS 
       [0021]    Biologically-based photoluminescent chemosensors (biosensors) have been proposed that may be interrogated optically. Each such sensor typically has an optode coupled to a fluorescence chemosensor element. This interrogation is performed by providing stimulus light through the optode and observing returned light through the optode at one or more fluorescent wavelengths, light at fluorescent wavelengths may be emitted by either fluorescence or phosphorescence in the associated chemosensor element. These sensors include sensors having one or more biocomponents comprising a dehalogenase, a hydrolase, a lyase and/or an oxygenase enzyme immobilized and/or stabilized in the sensor. These biosensors may monitor or detect the presence and/or concentration of one or more analytes, such as hydrocarbons and alcohols, polycyclic hydrocarbons, s-triazines, chlorinated ethenes, orthosulfates, organophosphates, or amine-substituted chemicals; this is not intended to be a complete list. S-triazines include, for example, the chlorinated herbicide atrazine, simazine, terbutylazine, propazine, cyanazine, diethylatrazine and deisopropylatrazine, plus other s-triazines, melamine, lindane and DDT. Chlorinated ethenes include, for example, tetrachloroethene (a.k.a., perchloroethene (PCE)), trichloroethene (TCE), dichloroethene isomers and vinyl chloride (VC). Organophosphates include, for example, the pesticides methyl parathion, chlorpyrifos, and diazinon; the nerve agent sarin; and solvents and plasticizers such as tributylphosphate, tris(2-ethylhexyl)phosphate and triphenyl phosphate. Organosulfates include, for example, cerebroside-3-sulfate, phenol sulfates, chondroitin sulfate, karatan sulfate, dermatan sulfate, choline sulfate, polysulfates of cellulose, heparitin sulfate, heparan sulfate, and heparin. Amine-substituted chemicals include, for example, melamine, ammeline, ammelide, serine, biotin, and aniline. There are many other such chemicals that may be of interest. 
         [0022]    Enzyme-based biosensors have been developed for ethanol that use alcohol oxidase, which catalyzes the reaction of ethanol and oxygen to form acetaldehyde and hydrogen peroxide. Mitsubayashi et al. described an optical biosensor in which alcohol oxidase was immobilized on the tip of a fiber optic oxygen sensor that used a photoluminescent ruthenium complex. This biosensor was found to detect ethanol in aqueous solutions in the range 0.5-9 millimolar, and was also effective in gaseous samples with ethanol concentrations from 0.7 to 50 ppm. Other researchers used coimmobilized alcohol oxidase and horseradish peroxidase, immobilized on an optical oxygen sensor, to measure methanol in n-hexane in the range 2-90 millimolar. 
         [0023]    In addition to chemosensor elements based on enzymatic oxidation with measurement of oxygen consumption as described in the background, other biologically based, optically-read, chemosensor components may incorporate a culture of living microorganisms to provide cofactors such as NADH or to permit detection based on gene expression. Although these sensors require frequent servicing to maintain or replace the cultures, and are a bit slower to react, such biosensors may be prepared for the detection of many hydrocarbons. Detection of various aromatic compounds at approximately 1 millimolar was achieved by Thavarungkul et al. using a culture of  Pseudomonas cepacia , Rella et al. used  Bacillus stearothermophilus  in a hydroxyethyl methacrylate membrane to measure phenol, catechol, and related compounds. Optically-read biosensors for the measurement of toluene using whole cells expressing toluene o-monooxygenase have resolved 0.3 milligrams per liter. An enzyme-based biosensor embodying a layer containing living cells and other components as an chemosensor element has been demonstrated for dichloroethane in water, as well as atrazine, lindane, and chlorohexane. It has been proposed that such sensors could be lyophilized for storage, and rehydrated before use. 
         [0024]    Antibodies to particular analytes have also been used to bind photoluminescent analytes at sensor tips. Chemosensor elements embodying such antibodies may also be used in the system as herein described. 
         [0025]    Fluorescent reagents that may be embodied in a biological or non-biological chemosensor include trisodium 8-hydroxy-1,3,6-trisulphonate for pH sensors, fluoro (8-anilino-1-naphthalene sulphonate) for Na +  ion sensors, and acridinium- and quinidinium-based reagents for halide sensors. 
         [0026]    A system  200  ( FIG. 2 ) using one or more sensors has several chemosensor components  202 ,  204 ,  206 , each of which is suitable for monitoring or detecting one or more analytes of interest and which provides fluorescence that varies with the analyte concentration. 
         [0027]    Each of these sensor elements is coupled to receive stimulus light from a separate interrogation light source  208 ,  210 ,  212 , coupled directly or through a stimulus optical fiber to chemosensor component  202 ,  204 ,  206 . For simplicity, any wavelength-selective devices necessary to exclude light at fluorescence wavelength are not shown separately in  FIG. 2  and are presumed to be included in the light source  208 ,  210 ,  212  although in an alternative embodiment multiple light sources  208 ,  210 ,  212  may exclude light at fluorescent wavelength by using separate light paths through a single wavelength-selective device. Interrogation light source  208 ,  210 ,  212  incorporates a light emitting device, which in an embodiment is a pulsed or modulated laser, and in an alternate embodiment is a pulsed or modulated light emitting diode. 
         [0028]    Each sensor element  202 ,  204 ,  206  is coupled to pass emitted fluorescent light through a sensing optical fiber to a common wavelength-selective device  214 , which may be a filter. Light passing through wavelength-selective device  214  continues to a common photodetector  216 . Photodetector  216  in an embodiment comprises a photomultiplier tube, in an alternate embodiment photodetector  216  is based on a P-Intrisic-N (PIN) diode. In other embodiments photodetector  216  is based on such other photodetector as is appropriate for detecting light of the fluorescent wavelength. In an alternate embodiment fluorescent from sensor elements  202 ,  204 ,  206  is coupled to a wavelength selective device  214  along separate optical paths or separate optical fibers and corresponding separate optical paths emerging from wavelength selective device  214  to converge on a photodetector  216 . 
         [0029]    Light sources  208 ,  210 ,  212  operate independently under control of a processing device  218 , which may be a computer or may be a microcontroller such as a Microchip PIC-16, a Motorola 6811, or an Intel 8096 or 8051 family member such as are equipped with an analog-to-digital converter, or may readily communicate with an analog-to-digital converter. 
         [0030]    The connections of optical fibers carrying emitted fluorescent light to wavelength-selective device  214  are arranged such that light at stimulus wavelength is substantially unable to pass from one fiber into another. 
         [0031]    During operation, system  200  operates according to a time division multiplexing scheme as illustrated in  FIG. 3 . A repeated cycle occurs in which a first of the light sources  208  is activated by processing device  218  for a dwell time TDW to stimulate fluorescence in a first of the sensors  202 . After the dwell time, the processing device uses photodetector  216  to measure light at fluorescent wavelength; this light is received primarily from the first sensor  202  and is measured as R 202  in following equations. Next, first light source  208  is turned off and a second of the light sources  210  is activated by processing device  218  for dwell time TDW 2  to stimulate fluorescence in a second of the sensors  204 . In embodiments where each sensor is of the same type, each dwell time TDW, TDW 2  is the same, where sensors have different decay times the dwell times TDW, TDW 2  may be determined as appropriate to allow decay of fluorescence in the preceding sensor and adequate stimulation time for a reading. After the dwell time, the processing device uses photodetector  216  to measure light at fluorescent wavelength; this light is received primarily from second sensor  204  and is measured as R 204  in the following equations. Next, the second light source  210  is turned off and a third of the light sources  212  is activated by processing device  218  for the dwell time to stimulate fluorescence in a third of the sensors  206 . After the dwell time, the processing device uses its analog-to-digital converter and photodetector  216  to measure light at fluorescent wavelength; this light is received primarily from the third sensor  206 , and is measured as R 206  in following equations, and the third light source  212  is turned off. After any other light sources and detectors are driven, the cycle repeats. Light sources, for example  208  and  210 , may overlap briefly when transitioning from one light source to the next light source if convenient and primarily one light source is on when the corresponding sensor is read. 
         [0032]    After each measurement of light at stimulus wavelength, processing device  218  applies calibration and correction factors from calibration tables  220  and in an embodiment provides calibrated sensor data to a host system. In an alternative embodiment, processing device  218  compares calibrated sensor data to detection thresholds and activates appropriate warning devices (not shown). 
         [0033]    While the system has been illustrated with three sources and three sensors, in principle any number greater than or equal to two each of sources and sensors may be used. 
         [0034]    In an embodiment, all sensors are identical and monitor the same analyte at different locations. Since optical fibers, such as fibers  222 ,  224 , are available with low attenuation, and minor attenuation can be adjusted for in calibration tables  220  ( FIG. 2 ), sensors such as sensor  204  may be located one hundred or more meters from remaining components, such as photodetector  216 , of the system; this permits monitoring multiple locations within the same industrial plant, or contaminated site undergoing environmental remediation, using a common photodetector  216  and processing device  218 . 
         [0035]    In an alternative embodiment, each sensor  202 ,  204 ,  206  is sensitive to a different analyte, permitting use of a common photodetector  216  to continuously measure contamination by several different analytes. For example, system  200  may monitor a sewage treatment plant or a water treatment plant for several different substances in source water. 
         [0036]    Many fluorescent materials, including those in sensors as herein described, have an afterglow  252  as illustrated in  FIG. 4 . The intensity of light emitted at fluorescent wavelength decays with time after the stimulus source is turned off (e.g. at TA), typically following a decay curve that can be expressed as the sum of one or more exponential decay curves. In an embodiment, the difference between times TA and TB is a time constant TC of a predominant component of the decay curve. 
         [0037]    In a slow embodiment sensors have a short TC compared to dwell time TDW. In this embodiment, TDW is chosen to be a large enough multiple of time constant TC to prevent undue interference between sensor readings. 
         [0038]    In a high speed embodiment, each sensor reading is corrected according to a known decay curve of the sensor or sensors last measured before it in the time-division multiplexing scheme. For example, in a system operating according to the cycle described above with reference to  FIG. 3 , the corrections are performed as: 
         [0000]      Corrected 202 reading=R202−K1(206)*R206−K2(204)*R204 
         [0000]      Corrected 204 reading=R204−K1(202)*R202−K2(206)*R206 
         [0000]      Corrected 206 reading=R206−K1(204)*R204−K2(202)*R202 
         [0039]    In an alternative embodiment, as illustrated in  FIG. 5 , a stimulus light, such as light  208 , is turned on for a dwell time TDW, and light at fluorescent wavelength is measured at time  260  immediately prior to turnoff. In this embodiment, decay time is measured by measuring light received by photodetector  216  at fluorescent wavelength at several decay sampling times  262 ,  264 ,  266 . In order to prevent crosstalk, in a version of this embodiment a delay of several time constants is allowed before turning on stimulus light  210  coupled to the next sensor to be read in sequence within the cycle. The decrease in light received by photodetector  216  at times  262 ,  264 ,  266 , and the magnitude of the steady-state photoluminescence at time  260  are used to determine decay rate of the fluorescence. This decay rate is then used to compute a measurement of analyte concentration present at the sensor. In another embodiment, reading immediately prior to turn off  260 ,  270 , is omitted and decay rate is determined only from readings at times  262 ,  264 ,  266  taken within several time constants after the stimulus light is turned off. 
         [0040]    In an alternative embodiment, once decaying fluorescent light at sufficient sample times  262 ,  264 ,  266  is measured to compute the decay rate of the fluorescence, stimulus light  210  coupled to the next sensor to be read in sequence within the cycle is activated. The decay rate of fluorescence from the first sensor  202  is extrapolated to provide values for removing crosstalk by subtracting decaying fluorescence from sensor  202  from samples taken at times  270 ,  272 ,  274 ,  276  and containing information primarily from sensor  204 . 
         [0041]    In an alternative embodiment, wavelength selective device  214  is a prism or diffraction grating, and photodetector  216  has an array of two or more photosensitive elements. In this embodiment, photodetector  216  provides information regarding spectra of light received from each sensor. 
         [0042]    In an alternative embodiment, as illustrated in  FIG. 6 , each stimulus light source  302 ,  304 ,  306  couples through stimulus optical fibers to not one, but two, chemosensors  308 ,  310 ,  312 ,  314 ,  316 ,  318 . In this embodiment, as in the embodiment of  FIG. 2 , multiple chemosensors couple through sensing optical fibers through each wavelength selective device  320 ,  322 . Each wavelength selective device  320 ,  322 , couples to a photodetector  324 ,  326 . In this embodiment, as in the embodiment of  FIG. 2 , each chemosensor receives light from only one of the light sources  302 ,  304 ,  306 , and each chemosensor, such as chemosensor  308 , couples to only one wavelength selective device  320 ,  322 . Sensors coupling to the first wavelength selective device  320  are referenced as those of a first sensor group, and sensors coupling to the second selective device  322  are referred to as those of a second sensor group. 
         [0043]    In order to prevent crosstalk between sensors of the first sensor group and sensors of the second sensor group, a fluorescence-wavelength blocking device  332 , such as an optical filter, is provided at a stimulus-fiber connection of each optode, for preventing emitted light from passing through stimulus-fibers into other sensors and being picked up by their sensing optical fibers. 
         [0044]    In an alternative embodiment, a wavelength-selective device such as wavelength selective device  334 , which may be a filter, having a single input from the associated light source  302  and multiple outputs connected through separate stimulus fibers to each associated chemosensor  308 ,  314 , serves to permit stimulus wavelength light to enter each stimulus fiber while blocking light, including crosstalk light, at fluorescent wavelength. 
         [0045]    In all embodiments, processing device  328 ,  218 , provides calibrated sensor readings derived from reading the biosensors or chemosensors to a host, or compares readings against warning limits and activates warning devices, in a manner such that each sensor reading is clearly identified to the associated sensor. 
         [0046]    It is anticipated that the system as herein described, when equipped with appropriate chemosensor elements, is of use as an environmental monitoring system in the following fields: 
         [0047]    Water treatment process monitoring. In both drinking water and wastewater treatment processes it is desirable to monitor contaminant levels for the protection of human and environmental health. Given the high flow rates of these processes, continuous monitoring of specific chemicals is desirable. 
         [0048]    Protection from chemical terrorism of water supplies. The possibility of terrorist attacks by the addition of toxic chemicals to water supplies has arisen in recent years. Devices capable of continuous monitoring for multiple toxic analytes at low levels are of particular interest to detect such chemicals. 
         [0049]    Monitoring of remediation processes. Once a remediation process has been designed and implemented at a contaminated site, its effectiveness must be established through a program of periodic monitoring, often at more than one location on the site. Such monitoring can be performed with a monitoring system as herein described. 
         [0050]    Environmental monitoring. It is often desirable to monitor sensitive water sources (ground water wells, rivers, lakes, etc.) that are downgradient from industrial sites and other sources of contaminants that may leak or spill. 
         [0051]    Precision agriculture. The goal of precision agriculture is to apply the correct amount of fertilizer and pesticide on every portion of a field, recognizing that different amounts are required depending on slope, exposure, soil type, and other factors. Multiple chemosensors may be implanted in a field and coupled by optical fiber to a common photodetector at a central monitoring point in the field. 
         [0052]    Changes may be made in the above methods and systems without departing from the scope hereof. It should thus be noted that the matter contained in the above description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of the present method and system and reasonable variations thereof, which might be said to fall therebetween.