Patent Publication Number: US-2023158497-A1

Title: Air to liquid micro-fluidic chamber

Description:
CROSS-REFERENCE TO RELATED APPLICATION(S) 
     This application is a division of U.S. application Ser. No. 16/379,890 filed Apr. 10, 2019 and issued as U.S. Pat. No. 11,548,001 on Jan. 10, 2023, which claims the benefit of and priority to U.S. Provisional Patent Application No. 62/655,478 filed on Apr. 10, 2018. The specifications of these applications are incorporated herein by reference, each in its entirety. 
    
    
     GOVERNMENT INTEREST 
     The invention described herein may be manufactured and used by or for the Government of the United States for all government purposes without the payment of any royalty. 
    
    
     BACKGROUND 
     Field of the Invention 
     The embodiments herein generally relate to contaminant detectors, and more particularly to detectors of organo-phosphate compounds, opioids, cannabinoids, and other classes of organic compounds. 
     Background of the Invention 
     There is a gap in surveillance in military and civilian operations. For example, there is concern with the air quality in aircraft for exposure to organo-phosphates that are found in fluids used by aircraft; specifically, tri-cresyl phosphates. There have been numerous attempts to measure these substances, but the methodology generally requires sampling and submission to a laboratory, which does not permit real-time analysis. Furthermore, the existing methodologies typically require substantial dilutions of the samples that elevates the detection limit to μg/sample. Another group of toxic organo-phosphates are pesticides and nerve agents. Due to the extreme toxicity of these agents it would be desirable to detect the substances at the femtogram level, and develop equipment that can operate autonomously and record data in real-time. 
     BRIEF SUMMARY OF THE INVENTION 
     In view of the foregoing, an embodiment herein provides a system comprising a pump to deliver vapor comprising airborne contaminants comprising organic compounds comprising a target analyte; a collector to transfer the airborne contaminants by autonomous liquid extraction into a mobile organic liquid phase; a micro-fluidic chamber comprising immobilized biorecognition elements that bind to analytes delivered from the mobile organic liquid phase; a mechanism to introduce the mobile organic liquid phase to a buffer containing a plurality of substrates causing a series of biochemical reactions that create a change corresponding to a concentration of the target analyte; and a detector to perform real-time analysis that correlates to a concentration of the organic compounds to determine a presence of the target analyte. 
     The collector may comprise a tube comprising silica gel coated with a xerogel to collect the organic compounds. The collector may comprise a microelectromechanical systems (MEMS) device to collect the organic compounds. The system may comprise a reservoir to hold the mobile organic liquid phase; and a valve to control delivery of the mobile organic liquid phase into the collector, wherein the mobile organic liquid phase comprises a non-polar solvent. The non-polar solvent may comprise hexane. The system may comprise an alarm that is triggered upon detection of the presence of the target analyte above a predetermined level. 
     Another embodiment provides an apparatus comprising a collector to collect a plurality of organic classes of compounds from an air sample; a micro-fluidic chamber to combine the collected plurality of organic classes of compounds with a biochemically and chemically reactive mobile phase analytic solution; and a detector to detect a presence of phosphate compounds in the mobile phase analytic solution. The apparatus may comprise a column containing a xerogel coated silica gel to collect the plurality of organic classes of compounds; and a heater to heat the column. 
     The apparatus may comprise a MEMS device to collect the plurality of organic classes of compounds. The apparatus may comprise a microchannel reactor comprising immobilized acetylcholine esterase that is introduced to the collected plurality of organic classes of compounds and the mobile phase analytic solution. The detector may comprise a buffer to detect acetylcholine esterase activity and trigger a fluorescent-producing reaction and create a fluorophore upon detecting a presence of acetylcholine esterase that is inhibited by the phosphate compounds in the mobile phase analytic solution. The apparatus may comprise a light source to excite the fluorophore. The apparatus may comprise a photodiode to measure the fluorophore. The microchannel reactor may comprise a liquid chamber containing a drop-wise addition channel. 
     Another embodiment provides a method comprising converting acetylcholine to acetate and choline; oxidizing the choline to derive betaine aldehyde and hydrogen peroxide; oxidizing the hydrogen peroxide to generate a hydrogen ion and electron; and generating a fluorophore from the hydrogen ion and electron. The method may comprise introducing an enzyme comprising any of acetylcholine esterase, cyclooxygenase, and horseradish peroxidase to the acetylcholine to convert the acetylcholine to acetate and choline. The method may comprise immobilizing the enzyme. The method may comprise oxidizing the hydrogen peroxide by a peroxidase. The method may comprise exciting the fluorophore to fluorescence. The method may comprise correlating the fluorescence to an inverse correlation of a concentration of organo-phosphates. 
     These and other aspects of the embodiments herein will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following descriptions, while indicating preferred embodiments and numerous specific details thereof, are given by way of illustration and not of limitation. Many changes and modifications may be made within the scope of the embodiments herein without departing from the spirit thereof, and the embodiments herein include all such modifications. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The embodiments herein will be better understood from the following detailed description with reference to the drawings, in which: 
         FIG.  1    is a block diagram illustrating a gas-to-liquid transfer system, according to an embodiment herein; 
         FIG.  2 A  is a block diagram illustrating the collector of the gas-to-liquid transfer system of  FIG.  1    with a tube, according to an embodiment herein; 
         FIG.  2 B  is a block diagram illustrating the collector of the gas-to-liquid transfer system of  FIG.  1    with a MEMS device, according to an embodiment herein; 
         FIG.  3    is a block diagram illustrating the gas-to-liquid transfer system of  FIG.  1    with a reservoir and valve, according to an embodiment herein; 
         FIG.  4    is a block diagram illustrating the gas-to-liquid transfer system of  FIG.  1    with an alarm, according to an embodiment herein; 
         FIG.  5    is a block diagram illustrating an apparatus to perform gas-to-liquid transfer, according to an embodiment herein; 
         FIG.  6 A  is a block diagram illustrating the apparatus of  FIG.  5    with a column and heater, according to an embodiment herein; 
         FIG.  6 B  is a block diagram illustrating the apparatus of  FIG.  5    with a MEMS device, according to an embodiment herein; 
         FIG.  7    is a block diagram illustrating the apparatus of  FIG.  5    with a microchannel reactor, according to an embodiment herein; 
         FIG.  8    is a block diagram illustrating aspects of the detector of the apparatus of  FIG.  5   , according to an embodiment herein; 
         FIG.  9    is a block diagram illustrating the apparatus of  FIG.  5    with a light source, according to an embodiment herein; 
         FIG.  10    is a block diagram illustrating the apparatus of  FIG.  5    with a photodiode, according to an embodiment herein; 
         FIG.  11    is a block diagram illustrating aspects of the microchannel reactor of the apparatus of  FIG.  5   , according to an embodiment herein; 
         FIG.  12 A  is a flow diagram illustrating a method of performing a chemical process, according to an embodiment herein; 
         FIG.  12 B  is a flow diagram illustrating a method of introducing an enzyme in the chemical process of  FIG.  12 A , according to an embodiment herein; 
         FIG.  12 C  is a flow diagram illustrating a method of immobilizing an enzyme in the chemical process of  FIG.  12 B , according to an embodiment herein; 
         FIG.  12 D  is a flow diagram illustrating a method of oxidizing the hydrogen peroxide in the chemical process of  FIG.  12 A , according to an embodiment herein; 
         FIG.  12 E  is a flow diagram illustrating a method of exciting the fluorophore in the chemical process of  FIG.  12 A , according to an embodiment herein; 
         FIG.  12 F  is a flow diagram illustrating a method of correlating a fluorescence in the chemical process of  FIG.  12 A , according to an embodiment herein; 
         FIG.  13    is a schematic representation of the chemical process of the method of  FIG.  12 A , according to an embodiment herein; 
         FIG.  14 A  is a schematic diagram illustrating a microfluidic device, according to an embodiment herein; 
         FIG.  14 B  is a schematic diagram illustrating a chemical scheme of the enzyme immobilization structure, according to an embodiment herein; 
         FIG.  14 C  is a graphical representation that correlates the protein concentration as a function of flow rate, according to an embodiment herein; 
         FIG.  14 D  is a graphical representation that correlates the fluorescence intensity as a function of flow rate, according to an embodiment herein; 
         FIG.  15 A  is a graphical representation that illustrates the relative fluorophore intensity for various solvents, according to an embodiment herein; 
         FIG.  15 B  is a schematic diagram illustrating a microchannel reactor, according to an embodiment herein; 
         FIG.  16 A  is a schematic diagram illustrating a detector system, according to an embodiment herein; 
         FIG.  16 B  is a graphical representation that correlates the current (corresponding to the fluorescence) as a function of time, according to an embodiment herein; 
         FIG.  17    is another graphical representation that correlates the current (corresponding to the fluorescence) as a function of time, according to an embodiment herein; 
         FIG.  18    is a schematic diagram illustrating a gas-phase analyte to liquid-phase transfer system, according to an embodiment herein; 
         FIG.  19    is a schematic diagram illustrating a vapor generation and sample delivery system for characterizing a gas sample collector using a diffusion vial and sample loop, according to an embodiment herein; 
         FIG.  20    is a graphical representation that correlates the integrated area NPD signal from PC-released TBP as a function of collection time, according to an embodiment herein; 
         FIG.  21    is a schematic diagram illustrating a gas-to-liquid transfer system, according to an embodiment herein; 
         FIG.  22    is a graphical representation that correlates the concentration of TBP in hexane as a function of peak area, according to an embodiment herein; and 
         FIG.  23    is a schematic diagram illustrating a system for continuous operation of an air-to-liquid sampler, according to an embodiment herein. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Embodiments of the disclosed invention, its various features and the advantageous details thereof, are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted to not unnecessarily obscure what is being disclosed. Examples may be provided and when so provided are intended merely to facilitate an understanding of the ways in which the invention may be practiced and to further enable those of skill in the art to practice its various embodiments. Accordingly, examples should not be construed as limiting the scope of what is disclosed and otherwise claimed. 
     The embodiments herein provide a gas-to-liquid transfer system to detect the presence of contaminants such as organo-phosphate compounds, among others. A collector collects the air contaminants for transfer to a dual reaction liquid chamber liquid that adds a solvent, such as hexane, to the collected organo-phosphates. A detector is provided to detect the presence of phosphate compounds, for example, by introducing an enzyme such as acetylcholine esterase, cyclooxygenase, or horseradish peroxidase. The embodiments herein utilize fluorescence emissions or other detectors (i.e., electrochemical) to determine the presence of the contaminants. 
     Referring now to the drawings, and more particularly to  FIGS.  1  through  23   , where similar reference characters denote corresponding features consistently throughout, there are shown exemplary embodiments. In the drawings, the size and relative sizes of components, layers, and regions may be exaggerated for clarity. 
       FIG.  1    illustrates a system  10  comprising a pump  15  to deliver vapor  20  comprising airborne contaminants  25  comprising organic compounds  35  comprising a target analyte  30 . The system  10  may be a portable system that is sized to be placed and removed from aircraft or other environmental location where air detection is required. In some examples, the pump  15  may comprise any of an electrical pump, mechanical pump, pneumatic pump, and electro-mechanical pump, or combinations thereof. The airborne contaminants  25  may comprise a plurality of different types of contaminants. In an example, the organic compounds  35  may comprise any of organo-phosphate compounds, opioids, cannabinoids, and other classes of organic compounds. The target analyte  30  may be selected based on a predetermined classification of target analytes that are desired to be detected and analyzed. Accordingly, the target analyte  30  comprises a chemical signature that the system  10  is configured to detect and analyze based on predetermined and/or pre-programmed chemical signature properties that are to be compared against to determine if there is a match, and the level of contamination or concentration of the target analyte  30  in the vapor  20 . 
     The system  10  further comprises a collector  40  to transfer the airborne contaminants  25  by autonomous liquid extraction into a mobile organic liquid phase  45 . In an example, the collector  40  may comprise a gas-to-liquid collector that utilizes collection gels, beads, or other mechanisms to collect the airborne contaminants  25  for transfer by liquid extraction. The mobile organic liquid phase  45  may flow at any suitable rate necessary to carry the liquid phase of the airborne contaminants  25  and corresponding organic compounds  35 . In an example, the mobile organic liquid phase  45  may comprise a non-polar solvent  110 . For example, the non-polar solvent  110  may comprise hexane or any other suitable non-polar material. 
     The system  10  comprises a micro-fluidic chamber  50  comprising immobilized biorecognition elements  52  that bind to analytes  46  delivered from the mobile organic liquid phase  45 . For example, the biorecognition elements  52  may be natural or synthetic. In some examples, the biorecognition elements  52  may include antibody enzymes, nucleic acid aptamers, or molecularly imprinted polymers. Unbound biorecognition elements  52  (i.e., free of the target analyte  30 ) compared to biorecognition elements  52  bound with target analytes  30  enable detection. According to the embodiments herein, reagents (not shown) are added in an aqueous phase (e.g., oil in water droplet) to trigger reactions needed for detection of the target analyte  30 . For example, the reagents may include Amplex® red reagent, among others. The micro-fluidic chamber  50  may contain a micro-preconcentrator (μ-PC) device (not shown in  FIG.  1   ) to control the concentration levels of the organic compounds  35  to be utilized for testing and analysis in the system  10 . For example, the micro-fluidic chamber  50  and the μ-PC device may be used to increase the concentration of the organic compounds  35  (and thus, the target analytes  30 ) from a lower concentration to a higher concentration. 
     The system  10  may comprise a mechanism  55  to introduce the mobile organic liquid phase  45  to a buffer  60  containing a plurality of substrates  65  causing a series of biochemical reactions that create a change corresponding to a concentration of the target analyte  30 . In an example, the mobile organic liquid phase  45  may be introduced by drop-wise addition, and thus the mechanism  55  may be any suitable device, such as a mechanical dropper, that transfers the mobile organic liquid phase  45  to the buffer  60 . The buffer  60  may be any suitable type of buffering solution to control some of the chemical properties associated with the mobile organic liquid phase  45 , such as the acidity, etc. For example, the buffer  60  may comprise any of acetylcholine esterase, cyclooxygenase, and horseradish peroxidase reacting with the acetylcholine to convert the acetylcholine to acetate and choline. The plurality of substrates  65  may comprise any suitable type of chemical material or molecules upon which the enzymes in the mobile organic liquid phase  45  act including bonding. The system  10  further comprises a detector  70  to perform real-time analysis  75  that correlates to a concentration of the organic compounds  35  to determine a presence of the target analyte  30 . In an example, the detector  70  may comprise a photodetector configured to detect of fluorescence emission. Moreover, the detector  70  may comprise any of an electronic, electrochemical, plasmonic, surface plasmon resonance, absorption, emission, electron transfer, and charge transfer device, or combinations thereof, or any other suitable detection system. The detector  70  may comprise a processor or may be communicatively linked to a processor, computer, or server device(s) to perform the real-time analysis  75  of the organic compounds  35  in order to determine the presence of the target analyte  30 . 
     In an alternative embodiment, instead of enzymes generating a read-out, the analytes of interest (e.g., target analyte  30 ) can interact with the biorecognition elements  52 , freeing them from the surface of a microreactor. These biorecognition elements  52  can then be detected downstream using the detector  70  with or without capture. 
       FIG.  2 A , with reference to  FIG.  1   , illustrates that the collector  40  may comprise a tube  80  comprising silica gel  85  coated with a xerogel  90  to collect the organic compounds  35 . The tube  80  may comprise any suitable size or material such as glass or durable plastic, for example. In an example, the silica gel  85  coated with the xerogel  90  may comprise solid pellets that are positioned in the tube  80 . In another example, the silica gel  85  may comprise viscous gel or material with the xerogel  90  coated thereon, and which are positioned in the tube  80 . Accordingly, the embodiments herein are not restricted to any particular configuration of the silica gel  85  and/or xerogel  90 . The  FIG.  2 B , with reference to  FIGS.  1  and  2 A , illustrates that the collector  40  may comprise a MEMS device  95  to collect the organic compounds  35 . A non-limiting example of a MEMS device/system that could be used for the MEMS device  95  herein is described in Bae, B., et al., “A Fully-Integrated MEMS Preconcentrator for Rapid Gas Sampling,” U.S. Air Force Report AFRL-PR-WP-TP-2007-224 for submission of a Conference paper submitted to the Proceedings of the Transducers 2007 Conference, Mar. 19, 2007, pp. 1-4, the complete disclosure of which, in its entirety, is herein incorporated by reference. 
       FIG.  3   , with reference to  FIGS.  1  through  2 B , illustrates that the system  10  may comprise a reservoir  100  to hold the mobile organic liquid phase  45 . The reservoir  100  may be any suitable type of liquid chromatographic instrument that may be configured to retain the mobile organic liquid phase  45 . In an example, the reservoir may be configured to contain pressurized solvent and may comprise vacuum connections for drawing the mobile organic liquid phase  45  therefrom and/or thereto. 
     The system  10  may comprise a valve  105  to control delivery of the mobile organic liquid phase  45  into the collector  40 . The valve  105  may comprise any suitable type of electrical, mechanical, pneumatic, or electro-mechanical valve, or combinations thereof. Moreover, the valve  105  may be automatically controlled by a local or remote processor or controller (not shown) or controlled by user intervention, as necessary.  FIG.  4   , with reference to  FIGS.  1  through  3   , illustrates that the system  10  may comprise an alarm  115  that is triggered upon detection of the presence of the target analyte  30  above a predetermined level. The alarm  115  may comprise any of an audio alarm, visual alarm, or combinations thereof. The alarm  115  may be communicatively coupled to the detector  70  and/or a processor or computer that transmits a signal to the alarm  115  upon the detector  70  detecting the presence of the target analyte  30  above a pre-programmed predetermined level. Since the detection and analysis of the presence of the target analyte  30  occurs in real-time, the alarm  115  is also triggered in real-time to alert users (i.e., pilots or other aircraft personnel, for example) to exit the aircraft or otherwise take precautionary measures to protect themselves from a potentially toxic environment due to the presence of the target analyte  30 . Moreover, the alarm  115  may comprise variable outputs based on the level of the presence of the target analyte  30 . For example, a yellow light may be output to indicate the presence of the target analyte  30  at levels that are not considered immediate health threats. Alternatively, a red light may be output to indicate a significant health threat to personnel in the vicinity of the system  10  and vapor  20 . Similarly, the variable outputs may be audio-based with different pitches, tones, or frequencies depending on the level of the threat that is detected. In another example, the audio output from the alarm  115  may be a computer-generated voice providing instructions for personnel as well as outputting the type of target analyte  30  that has been detected as well as the level (i.e., amount) of detection. 
       FIG.  5   , with reference to  FIGS.  1  through  4   , illustrates another embodiment that provides an apparatus  150  comprising a collector  40  to collect a plurality of organic classes of compounds  155  from an air sample  160 . The apparatus  150  may be miniaturized to be portable and easily transferred from various settings such as aircraft, for example. The plurality of organic classes of compounds  155  may comprise any of organo-phosphate compounds, opioids, cannabinoids, and other classes of organic compounds, according to some examples. The air sample  160  may be the ambient air in an aircraft cockpit, for example. However, the air sample  160  and corresponding environmental location for using the apparatus  150  may be any suitable location where the detection and analysis of the air sample  160  is required. 
     The apparatus  150  comprises a micro-fluidic chamber  50  to combine the collected plurality of organic classes of compounds  155  with a biochemically and chemically reactive mobile phase analytic solution  170  (e.g., which may be the buffer  60  containing the plurality of substrates  65 , for example). According to an example, single or multiple pairs of immiscible liquids may extract and transfer contaminants to the biochemically and chemically reactive mobile phase analytic solution  170 . The apparatus  150  further comprises a detector  70  to detect a presence of phosphate compounds  175  in the mobile phase analytic solution  170 . As such, the mobile phase analytic solution  170  may be any suitable type of buffering solution to control chemical properties, and may contain any suitable type of chemical material or molecules upon which enzymes act, including bonding. In an example, the mobile phase analytic solution  170  may comprise the mobile organic liquid phase  45 , described above, of the collected plurality of organic classes of compounds  155 . 
       FIG.  6 A , with reference to  FIGS.  1  through  5   , illustrates that the apparatus  150  may comprise a column  180  containing a xerogel  90  coated silica gel  85  to collect the plurality of organic classes of compounds  155 . The column  180  may comprise any suitable size or material such as glass or durable plastic, for example. In an example, the silica gel  85  is coated with the xerogel  90  and may comprise solid pellets that are positioned in the column  180 . In another example, the silica gel  85  may comprise viscous gel or material with the xerogel  90  coated thereon, and which are positioned in the column  180 . According to an example, the column  180  may be configured to withstand pressured liquids including the mobile phase analytic solution  170 , the mobile organic liquid phase  45 , or other liquids, solvents, or solutions. The apparatus  150  may comprise a heater  185  to heat the column  180 . The heater  185  may comprise any suitable type of heater  185  that can be automatically and/or manually controlled to heat the column  180  to desired temperatures. In an example, the heater  185  may be used to heat to the column  180  to help facilitate a separation process of the mobile phase analytic solution  170 . 
       FIG.  6 B , with reference to  FIGS.  1  through  6 A , illustrates that the apparatus  150  may comprise a MEMS device  95  to collect the plurality of organic classes of compounds  155 .  FIG.  7   , with reference to  FIGS.  1  through  6 B , illustrates that the apparatus  150  may comprise a microchannel reactor  190  comprising immobilized acetylcholine esterase  195  or other alternative selective enzyme or aptamer that is inhibited by the phosphate compounds  175  that is introduced to the collected plurality of organic classes of compounds  155  and the mobile phase analytic solution  170 . According to an example, the microchannel reactor  190  may comprise a continuous plug flow reactor, although other types of reactors may be utilized in accordance with the embodiments herein. The microchannel reactor  190  may comprise a series of serpentine channels in which the chemical reactions of the mobile phase analytic solution  170  occur. Moreover, the microchannel reactor  190  may be configured to have suitable heat exchange thermal properties to withstand exothermic reactions of the mobile phase analytic solution  170 . 
       FIG.  8   , with reference to  FIGS.  1  through  7   , illustrates that the detector  70  may comprise a buffer  60 . As described above, and in some examples, the buffer  60  may comprise any of acetylcholine esterase, cyclooxygenase, and horseradish peroxidase reacting with the acetylcholine to convert the acetylcholine to acetate and choline. The buffer  60  is to detect acetylcholine esterase activity and trigger a fluorescent-producing reaction and create a fluorophore  200  upon detecting a presence of acetylcholine esterase  195  that is inhibited by the phosphate compounds  175  in the mobile phase analytic solution  170  resulting in a quenching of the enzyme comprising any of the acetylcholine esterase, cyclooxygenase, and horseradish peroxidase. The fluorophore  200  may be any suitable type of fluorescent chemical compound capable of re-emitting light upon excitation caused by the reaction of the buffer  60 . 
       FIG.  9   , with reference to  FIGS.  1  through  8   , illustrates that apparatus  150  may comprise a light source  205  to excite the fluorophore  200 . In an example, the light source  205  may comprise a light-emitting diode (LED) or any other suitable light generating and emitting device.  FIG.  10   , with reference to  FIGS.  1  through  9   , illustrates that apparatus  150  may comprise a photodiode  210  to measure the fluorophore  200 . The photodiode  210  may be configured to measure the light emitted by the fluorophore  200  and convert the light into an electrical current. According to an example, the concentration of the phosphate compounds  175  is inversely proportional to the excitation of the fluorophore  200 , which indicates the presence of the toxins (based on a predetermined chemical signature as described above) from the plurality of organic classes of compounds  155  in the air sample  160 .  FIG.  11   , with reference to  FIGS.  1  through  10   , illustrates that microchannel reactor  190  may comprise a liquid chamber  215  containing a drop-wise addition channel  220 , according to an example. The liquid chamber  215  and drop-wise addition channel  220  may be any suitable devices and instruments capable of retaining and transferring the aforementioned solvents and solutions used in the apparatus  150 . 
       FIG.  12 A , with reference to  FIGS.  1  through  11   , is a flow diagram illustrating a method  300  according to an embodiment herein.  FIG.  13    illustrates a schematic representation of the chemical process described by the method  300 . According to an example, the method  300  comprises converting ( 305 ) acetylcholine to acetate and choline; oxidizing ( 310 ) the choline to derive betaine aldehyde and hydrogen peroxide; oxidizing ( 315 ) the hydrogen peroxide to generate a hydrogen ion and electron; and generating ( 320 ) a fluorophore  200  from the hydrogen ion and electron.  FIG.  12 B , with reference to  FIGS.  1  through  12 A , is a flow diagram illustrating that the method  300  may comprise introducing ( 325 ) an enzyme comprising any of acetylcholine esterase, cyclooxygenase, and horseradish peroxidase to the acetylcholine to convert the acetylcholine to acetate and choline.  FIG.  12 C , with reference to  FIGS.  1  through  12 B , is a flow diagram illustrating that the method  300  may comprise immobilizing ( 330 ) the enzyme.  FIG.  12 D , with reference to  FIGS.  1  through  12 C , is a flow diagram illustrating that the method  300  may comprise oxidizing ( 335 ) the hydrogen peroxide by a peroxidase.  FIG.  12 E , with reference to  FIGS.  1  through  12 D , is a flow diagram illustrating that the method  300  may comprise exciting ( 340 ) the fluorophore  200  to fluorescence.  FIG.  12 F , with reference to  FIGS.  1  through  12 E , is a flow diagram illustrating that the method  300  may comprise correlating ( 345 ) the fluorescence to an inverse correlation of a concentration of organo-phosphates. 
     AChE is an important enzyme that breaks down acetylcholine, a key substance as a neurotransmitter. Organo-phosphates are toxic because they inhibit AChE activity by binding on the active site of AChE covalently. The embodiments herein provide an inhibition mechanism to detect the organo-phosphate in the air, as depicted in  FIG.  13   , with reference to  FIGS.  1  through  12 F . There are three reactions which are concerned with three enzymes of AChE, COX, and HRP. Reaction  1  is a degradation reaction that converts acetylcholine to acetate and choline with AChE. Reaction  2  is an oxidation reaction of choline in which betaine aldehyde and hydrogen peroxide are derived, and the hydrogen peroxide is oxidized by peroxidase to generate hydrogen ion and electron. The hydrogen and electron activate the fluorescence substrate. The fluorescence is excited at 550 nm and 600 nm light is emitted. 
     The embodiments herein were experimentally tested according to the following series of experiments. The specific devices, orientations, configurations, geometries, sizes, temperatures, timings, ratios, speeds, techniques, colors, and/or types and amounts of materials, etc. described in the experiments below are merely exemplary, and the embodiments herein are not restricted to any particular structure, property, technique, or material described below. Accordingly, the experiments are merely being presented to demonstrate the feasibility of the embodiments herein and are not meant to restrict how the invention may be practiced. 
     Experiments 
     The microchannel reactor  190  may comprise a serpentine microfluidic channel  401  as depicted in  FIG.  14 A , and which may be fabricated by soft-lithography processing. For example, photo-lithography is used to make the mold, and white polydimethylsiloxane (PDMS, 1:10 ratio of cross linker and monomer)  403  is cured on the mold at 70° C. after spin coating at 250 RPM. The microfluidic channel  401  formed by soft-lithography is then covered with a transparent slab of PDMS  403 . The surface of the microfluidic channel  401  is exposed to UV-plasma to generate hydroxyl groups on the surface, to allow functionalization with amine groups via (3-aminopropyl) triethoxylane (3-APTES). Acetone and 3-APTES are mixed at a 7:3 ratios, and injected into the microfluidic channel  401  at a 100 μL/min flow rate at approximately 60° C. for approximately 4 hours. After functionalization, a cross-linker is developed using glutaraldehyde at a 6:4 ratio of PBS (phosphate buffer saline) and glutaraldehyde mixture (glutaraldehyde 10% in PBS) and injected at ˜50 μL/min flow rate for approximately 4 hours. Finally, enzymes of acetylcholinesterase (AChE, ˜12.5 choline oxidase (COX, ˜12.5 μL/mL), and horseradish peroxidase (HRP, ˜5 μL/mL) are immobilized on the surface of the microfluidic channel  401 .  FIG.  14 B  shows the chemical scheme of the enzyme immobilization structure  407  on the PDMS  403 . The prepared microchannel reactor  190  is filled with PBS and stored at approximately 4° C. The chain reaction using the microchannel reactor  190  is performed with reactant mixtures of acetylcholine (˜100 μM) and fluorescence substance (˜5 μL) as the mixture is injected into the microchannel reactor  190  at ˜200 μL/min flow rate at approximately 30° C. Fluorescence from the reaction is excited at ˜550 nm wavelength (green light), and emitted at ˜600 nm (orange light). 
     The microchannel reactor  190  is prepared as all enzymes are immobilized on the surface of the channel  401 . The cross-linker quantity and enzyme binding amount are controlled by changing functionalization time and flow rate.  FIG.  14 C  depicts the flow rate results. High enzyme immobilization level leads to high enzyme activity in the microchannel reactor  190 . Excessively high activity, however, leads to saturation in the fluorescence intensity, and such high activity can dilute the effect of organo-phosphate inhibition. Such competitive inhibition is usually solved with excessive enzyme amount. Thus, an appropriate enzyme amount should be immobilized to for high sensitivity of organo-phosphate inhibition. As with the functionalization time, the quantity of cross-linker can be varied. For high sensitivity of inhibition response, the functionalization time is determined at over 2 hours. The flow rate for enzyme immobilization is ˜30 μL/min as denoted by the dashed box in  FIG.  14 C . If a high level of protein is immobilized, the sensitivity decreases. A flow rate of 30 μL/min yields an appropriate reaction rate. Fluorescence is very sensitive, and it shows reasonable intensity with μmon concentration level of acetylcholine. When the flow rate is ˜30 μL/min, the relative fluorescence intensity is ˜75% of saturated intensity as indicated in the results of  FIG.  14 D . 
     The collector  40  may be an air-to-liquid (gas-to-liquid) tributyl phosphate (TBP) collector  40 . The TBP collection may include a gas-to-liquid extractor that is utilized in a laboratory hood. A beaker containing ˜10 mL TBP is placed adjacent to a probe on the TBP collector  40 . The collector  40  sniffs TBP, and the membrane is rinsed with hexane. The TBP on the membrane dissolves in hexane, and finally the TBP can be collected in a 1 mL syringe for injection into the reaction-detection system. 
     The microchannel reactor  190  is experimentally tested with non-polar organic solvents. With respect to the gas-to-liquid TBP collector  40 , the elute of sensor system includes solvents. The reactant with fluorescence substrate is, however, a water-based solution. Thus, droplet generating microfluidics is introduced. As indicated in  FIG.  15 A , various solvents are experimentally considered in terms of conservation of enzyme in the microchannel reactor  190  activity as denoted by the relative fluorophore intensity. Hexane is experimentally determined to be an appropriate solvent for both the gas-to-liquid TPB collector  40  and the microchannel reactor  190 . The drop-wise addition channel  220  may also be embedded in the microchannel reactor  190 .  FIG.  15 B  shows an experimental configuration of the microchannel reactor  190 . Continuous flow reaction was conducted using the microchannel reactor  190  prepared with the optimum conditions of an enzyme immobilization and organic solvent/water hybrid flow system. As a result, the fluorescence is observed from a cuvette  209  (denoted in  FIG.  16 A ) in which the fluorescence substrate is connected with the product of the enzyme reaction to generate fluorescence luminescence. The fluorescence is observed at ˜600 nm wavelength, excited at 550 nm wavelength. In the test of the sensing system, an inhibition substrate (e.g., TBP) is used at each concentration. Reactant flow rate also affects the inhibition, and the flow rate is determined to be ˜50 μL/min. As the inhibitor solubilized flow passes into the microchannel reactor  190 , the fluorescent intensity is observed to decrease. The competitive inhibition of TBP is direct, and the signal of fluorescence changes instantly. 
     The schematic configuration of the detector  70  is shown in  FIG.  16 A . Various geometries may be considered for effective detection of fluorescence emission in the micro-channel reactor  190  and structures for direct lighting-detecting are determined. For fast detection of signal, the detector  70  size should be miniaturized to allow integration with the micro-fluidic chamber  50 . For example, millimeter-sized components are used. An excitation light source  205  to generate light  206  may comprise an LED  207  and a polarizing filter  208 . A cuvette  209  is positioned in the pathway of the light  206 . Green filters may be used to reject stray light and excitation light. As emission wavelengths from the microchannel reactor  190 , 570 nm, 590 nm, and 610 nm polarizing filters which reject each wavelength are tested, and the photodetector  70  is placed behind polarizing the filters  208  and are selected with suitable properties. 
     With respect to the data acquisition process, the current from the photodetector  70  is measured using a digital multi-meter (DMM)  204 . As the system is operated, the detector  70  determines the fluorescence intensity associated with continuous reactant flow. The continuous changes in current are recorded and stored in a computer  203 . The noise from the recorded data may be removed by adjusting a low pass digital filter as part of the data processing. 
     The fluorescence intensity is detected after emission of the light. The detector  70  mechanism is based on a fluorescence microscope. Green LEDs (550 nm) are selected and tested as excitation light sources with small sizes and strong output intensity. The photodetectors  70  should be sensitive at near red color light. The Polarizing filter  208  rejects all wavelengths below the emission wavelength. 570 nm and 590 nm filters are experimentally tested, and a high quality of wavelength rejection is realized with a 610 nm polarizing filter  208 . Emission of the fluorescence substrate is known to be 600 nm, but a small size of the detected signal creates challenges in eliminating effects of light scattering penetration of excitation light  206  through the polarizing filter  208 . Therefore, over 600 nm filters can be utilized. As the results of using a 610 nm filter  208 , a small portion of fluorescence emission is rejected but the fluorescence information is more clea, and robust. Based on these efforts, all components were assembled with poly(methyl methacrylate) (PMMA). A two-dimensional laser cut PMMA pieces are bonded with epoxy, and a black PDMS  202  is used to cover the surrounding detector  70  to prevent light penetration from outside. Also, an aluminum channel is tested. The design of both the PMMA and aluminum detection channels are determined after optimization of various geometries of the positions of the excitation light source  205  and photodetector  70 . When the detector  70  is prepared and integrated with the microchannel reactor  190 , fluorescence intensity can be monitored and captured with a computer  203 . The fluorescence information is collected by the detector  70 , and the photodetector portion of the detector  70  generates current as the fluorescence intensity in the microfluidics.  FIG.  16 B  summarizes the results. Time  0  is the starting point of inhibition and at ˜200 pM TBP, the fluorescence changes as shown in  FIG.  16 B . The current scale is around nA before optimization.  FIG.  16 B  shows results at the μA scale, which is significantly improved in terms of sensitivity and stability for actual use. 
     The developed sensing-detecting system should be integrated with the TBP collector  40 . The experimental organo-phosphate detection system was designed and tested at the bread board level. The normal state of the system shows normal reaction conditions in which the chain reaction is performed. The chain reaction leads to the production of hydrogen which activates the fluorescence substrate ( FIG.  13   ). The system shows fluorescence intensity corresponding to acetylcholine concentration, and the fluorescence intensity is affected by acetylcholine concentration and AChE activity. The system has continuous flow while it is operated. During flow operation, there is a constant amount of acetylcholine (10 mM) and fluorescence substrate (50 μl/mL). Thus, AChE activity affects the fluorescence intensity and if there is a strong inhibitor in the elute, AChE activity will be decreased. The normal state of the system, therefore, should show constant intensity of fluorescence. The alternating flow of hexane and reactant by the droplet generator for merging two flows is read out by the fluorescence detector  70  in  FIG.  16 A . Experimentally, a strong pink color indicated that the enzyme chain reactions are in the stationary phase. The TPB collector  40  is operated and TBP in the air could be collected and resolved into hexane. The system is a batch type and the TBP could be recovered by hexane flushing by two syringes. When the syringe with collected TBP is injected into the system, the reaction is inhibited. 
       FIG.  17    shows the results of all experimental system operations. The fluorescence detector  70  generates current as there is over 610 nm light in the detector channel, and the signal data is the current value. The reaction system was stationary phase by ˜400 sec. The constant fluorescence intensity reflects normal enzymatic activity. After 400 sec, as the TBP syringe is injected, the signal decreases which means that inhibition of AChE occurs by the TBP collected from air. The dashed adjoint line in  FIG.  17    is filtered data which results from low pass filter adjustment. The decreased rate can be calculated to derive TBP concentration. With different amounts of organo-phosphates, the level of decreasing rate will be different. A high amount of organo-phosphate would lead to a more rapid decrease in fluorescence intensity. The calibration of decrease rate should be conducted for practical use. It is noted that enzyme activity can deactivate naturally as it is used. The deactivation without the presence of organo-phosphates should be addressed to yield stable operation in practical conditions. The effect is expected not to be significant, and it would be a slight change of the intensity. Therefore, the system should have a reference reactor which only has the flow of the reactant and fluorescence substrate. The reference reactor would improve the sensitivity and reliability of the system. Also, an automated data processing system could be developed for practical use in situ. 
     An exemplary TBP detection system  500  that was experimentally tested is illustrated in  FIG.  18   . Generally, the system  500  comprises an AChE reservoir  502  operatively connected to an air-to-liquid micro-preconcentrator (μ-PC)  506 . An air sample inlet  508  inputs into the μ-PC  506  also. An air sample vacuum pump  510  is operatively connected to the μ-PC  506 . A microchannel reactor substrate  520  is operatively connected to the μ-PC  506 . The microchannel reactor  520  comprises a transparent substrate cover  514 , a LED light source  516 , and a charge-coupled device (CCD)  518  as a photodiode for sensing emitted light. An enzyme reservoir  504  and waste reservoir  522  are operatively connected to the microchannel reactor substrate  520 . A vacuum pump  512  for AChE transfer is operatively connected to the waste reservoir  522 . A low-concentration mix of TBP in air passes over the μ-PC  506  where TBP is collected. The μ-PC  506  rapidly heats to develop a concentrated pulse of TBP vapor in a small chamber that encloses the μ-PC  506 . A portion of the concentrated pulse accumulates in solvent on the opposite side of a membrane that forms one wall of the chamber. The elevated concentration and temperature enhance the transfer of TBP into solvent. Solvent with the collected TBP is transferred to the detector, where it is incubated with an immobilized acetylcholine esterase within a microchannel reactor  520 . Aqueous solution for detection of acetylcholine esterase activity then passes over the immobilized enzyme, triggering a reaction producing a fluorescent substrate only if acetylcholine esterase is present and uninhibited by organophosphates/nerve agents. The LED light source  516  at 550 nm excites the fluorophore, and the emission is measured at 600 nm on a photodiode (CCD)  518 . The percent reduction of fluorescence correlates with the concentration of TBP in the sample. 
     Hexane is brought into direct contact with the xerogel in the μ-PC  506  material. A pair of valves may be used to direct air and liquid flows during the sample collection cycle and the flush cycle where TBP is transferred to hexane. Between collection cycles, the μ-PC  506  material is heated and dried with air. 
     TBP is placed in a diffusion vial and set in a heated chamber with continuous 100 mL/min flow of zero air. The flux rate from the vial is determined gravimetrically by weekly weight measurements. With a D size diffusion vial with a 1-inch stem at ˜80° C., the flux rate is determined to be 317 ng/min. This correlates with the estimated 390-ng/min flux rate for this vial. Since the sweep gas is 100 mL/min, the maximum concentration that can be delivered is approximately 400 ppb. The system  500  has a 500-μL sample loop so 2-ng of sample can be delivered to the test chamber shown in  FIG.  19   . Sample loops of 100 μL and 10 μL can also be used, and the sample stream could also be diluted to deliver lower concentrations of TBP samples. 
     The vapor system  500  was used to test some of the air-to-liquid sample collector concepts, but testing was also conducted with headspace samples of TBP in an open container. To estimate the concentration in headspace air samples, the upper limit can be determined by comparing the vapor pressure of TBP at ambient temperatures to the ambient pressure. At 25° C. the vapor pressure of TBP is on the order of 0.00113-mmHg. During testing, the barometric pressure is nominally 640-mmHg, so the concentration in the headspace is approximately 1.8-ppm. However, the concentration could be at least an order of magnitude lower as indicated in Skene, W., et al. “Vapor Pressure of Tri-n-Butyl Phosphonate,” J. Chem. Eng., Data, 1995, 40, pp. 394-397, the complete disclosure of which, in its entirety, is herein incorporated by reference. Sampling headspace requires the dense vapor to travel up a container from the liquid surface and mix with the air. As a dense vapor, TBP would require a long equilibration time. Therefore, in working with headspace samples of TBP in air, it is estimated that the concentration is between 100 ppb and 1 ppm by volume. 
     The roll of the μ-PC  506  in the system  500  is to capture the TBP in the air and concentrate it for delivery to the liquid. A series of tests were conducted to quantify the TBP capture and release properties for the μ-PC  506 . The high-surface area μ-PC  506  is first exposed to the TPB stream and then the valve is switched to connect the μ-PC  506  to a nitrogen phosphorous detector (NPD). Voltage is applied to a heater on the μ-PC  506  to produce a concentrated plume on analyte on the NPD.  FIG.  20    shows that the integrated area NPD signal trace is proportional to the mass of TBP that flows to the detector. The mass collected is proportional to the collection time, at least up to 5 minutes. Further testing shows that the linearity falters at long collection times. At 15 minutes, the measured total peak area is about 75% of the linear predicted area. 
     The ability of the μ-PC  506  to retain a sample of TBP is also tested. Even with warm nitrogen blowing over the surface of the μ-PC  506  for 4 extra minutes, there is no apparent loss in captured TBP. Certainly, longer intervals and higher flushing volumes will remove the TBP, but the xerogel coating has a high affinity for TBP to prevent quick loss in the captured TBP. 
     To estimate concentration capabilities of the μ-PC  506 , the system is calibrated with a 500-μL sampling loop. This loop replaces the μ-PC  506  to introduce a known quantity of analyte to the NPD. The TBP sample is generated with a D-sized diffusion vial in an oven at 80° C. The flux rate of TBP is estimated to be 393 ng/min. The flow rate of the zero-air sweep gas through the diffusion vial chamber is measured with a bubble meter to be 55.1 mL/min. Based on these measurements, the concentration of TBP in the stream is 7.16 ng/mL, so a 500-μL sample loop would contain 3.58 ng of TBP. 
     Experimentally, it was determined that hexane could be flushed directly over the μ-PC  506  to remove concentrated TBP. An experimental apparatus shown in  FIG.  21    was developed where the preconcentrator is a bed of microbeads  550  that are coated with xerogel. The microbeads  550  are packed in a tube that is wrapped with a heater for drying the packed bed after the hexane extraction. Valves  552 ,  553  at each end of the tube isolate the air collection system from the hexane. 
     During the TBP collection cycle, the valves  552 ,  553  are energized and a pump  555  pulls TBP-contaminated air through the microbeads  550 . After collecting for a set amount of time, typically 10&#39;s of minutes, the pump  555  is switched off and the valves  552 ,  553  are de-energized. Hexane is introduced into the upstream side of the microbeads  550  using a 1-mL syringe  554 . The hexane is then withdrawn through the same port and transferred to a detector system. A second syringe of clean hexane is flushed in and out of the collection tube to remove TBP remaining on the microbeads  550 . The valves  552 ,  553  are then switched on, and the pump  555  and heaters are also switched on. The PC tube is heated to 60° C. to vaporize any remaining hexane. At this temperature, all hexane is removed in less than five minutes. 
     The micro-bead packed PC system is tested to determine the concentration of TBP in the solvent. The original calibration is performed with acetonitrile, but the calibration is repeated with the hexane solvent and NPD. Five concentrations are run from 2 μg TBP/Liter hexane to 1000 μg/L, and the correlation between the area of the TBP peak and the concentration is shown in  FIG.  22   . 
     Tests were performed where TBP in air was drawn into the collector from the headspace above the surface of liquid TBP. After a collection of 25 minutes, 1 mL of hexane is injected into the μ-PC  506  and withdrawn from the same port. When 1-4, of this sample is injected into the calibrated flame ionization detector (FID), the peak area corresponds to 4.8 ng of TBP in the sample. A second flush 1-mL of hexane in the μ-PC  506  shows that the concentration goes to 8.4 ng of TBP, but a third flush of hexane shows that the μ-PC  506  is clean. Similar tests are performed at 5 and 10-minute collect times. 
       FIG.  23    illustrates one exemplary approach for automating the collector and detector in the system. In this system shown in  FIG.  23   , pump  1  and valve  1  are energized to pull an air sample into the collector. Check valves at the inlet of the μ-PC trap only allows the flow to come from one port of a tee at the inlet. After the collection, pump  2  and valve  2  energize to flush hexane back through the μ-PC trap. A check valve at the inlet tee directs the flow to the detector system. After a couple of trap volumes of hexane is flushed through the μ-PC, valve  2  de-energizes, and air pushes remaining hexane out of the trap. Additionally, pump  1 , valve  1 , and the heater can be activated to dry the trap before the next collection cycle. 
     Accordingly, the experiments demonstrate an organo-phosphates detection system based on an inhibition mechanism for acetylcholinesterase. A microchannel reactor  190  with chain reaction to yield fluorescence is experimentally demonstrated and a detection sub-system is constructed to readout the fluorescence intensity. The gas-to-liquid TBP collector  40  gathers TBP in the air, and transfers it to the liquid (hexane), which is injected into the enzymatic reaction microchannel reactor  190 . The detector  70  allows sensing of changes in the reaction rate by measuring the inverse proportionality to organo-phosphate concentration to fluorescence. 
     The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the appended claims.