Patent Description:
Periodic soil testing is an important aspect of the agricultural arts. Test results provide valuable information on the chemical makeup of the soil such as plant-available nutrients and other important properties (e.g. levels of nitrogen, magnesium, phosphorous, potassium, pH, etc.) so that various amendments may be added to the soil to maximize the quality and quantity of crop production.

In some existing soil sampling processes, collected samples are dried, ground, water is added, and then filtered to obtain a soil slurry suitable for analysis. Extractant is added to the slurry to pull out plant available nutrients. The slurry is then filtered to produce a clear solution or supernatant which is mixed with a chemical reagent for further analysis.

<CIT> discloses an apparatus for performing a field assay of an analyte with a microfluid cartridge and a reader. The microfluidic cartridge has a closed microfluidic circuit for mixing and recirculating a fluid. The reader is selectively communicated with the microfluidic cartridge. The first manifold is configured to distribute fluid uniformly to a plurality of channels of a surface acoustic wave (SAW) detector. A second manifold coupled to a return line is configured to remove the fluid from the plurality of channels of the SAW detector.

Improvements in testing soil, vegetation, and manure are desired.

The present invention provides an automated computer-controlled sampling system and related methods for collecting, processing, and analyzing soil samples for various chemical properties such as plant available nutrients (hereafter referred to as a "soil sampling system"). The sampling system allows multiple samples to be processed and analyzed for different analytes (e.g. plant-available nutrients) and/or chemical properties (e.g. pH) in a simultaneous concurrent or semi-concurrent manner, and in relatively continuous and rapid succession. Advantageously, the system can process soil samples in the "as collected" condition without the drying and grinding steps previously described.

The present system generally includes a sample preparation sub-system which receives soil samples collected by a probe collection sub-system and produces a slurry (i.e. mixture of soil, vegetation, and/or manure and water) for further processing and chemical analysis, and a chemical analysis sub-system which receives and processes the prepared slurry samples from the sample preparation sub-system for quantification of the analytes and/or chemical properties of the sample. The described chemical analysis sub-system can be used to analyze soil, vegetation, and/or manure samples.

In one embodiment, the sample preparation system generally includes a mixer-filter apparatus which mixes the collected raw soil sample in the "as sampled" condition (e.g. undried and unground) with water to form a sample slurry. The mixer-filter apparatus then filters the slurry during its extraction from the apparatus for processing in the chemical analysis sub-system. The filter may be separate The chemical analysis sub-system processes the slurry and performs the general functions of extractant and color-changing reagent addition/mixing, centrifugating or filtering the slurry sample via microporous filter to yield a clear supernatant, and finally sensing or analysis for detection of the analytes and/or chemical properties such as via colorimetric analysis. In various embodiments, all or part of the chemical analysis sub-system may be incorporated into one or more microfluidic devices of suitable configuration.

Although the sampling systems (e.g. sample collection, preparation, and processing) may be described herein with respect to processing soil samples which represents one category of use for the disclosed embodiments, it is to be understood that the same systems including the apparatuses and related processes may further be used for processing other types of agricultural related samples including without limitation vegetation/plant, forage, manure, feed, milk, or other types of samples. The embodiments of the invention disclosed herein should therefore be considered broadly as an agricultural sampling system. Accordingly, the present invention is expressly not limited to use with processing and analyzing soil samples alone for chemical properties of interest.

The present invention will become more fully understood from the detailed description and the accompanying drawings, wherein like elements are labeled similarly and in which:.

All drawings are not necessarily to scale. Components numbered and appearing in one figure but appearing un-numbered in other figures are the same unless expressly noted otherwise. A reference herein to a figure by a whole figure number which may appear in multiple figures bearing the same whole number but with different alphabetical suffixes shall be construed as a general reference to all of those figures unless expressly noted otherwise.

The features and benefits of the invention are illustrated and described herein by reference to exemplary ("example") embodiments.

In the description of embodiments disclosed herein, any reference to direction or orientation is merely intended for convenience of description and is not intended in any way to limit the scope of the present invention. Relative terms such as "lower," "upper," "horizontal," "vertical,", "above," "below," "up," "down," "top" and "bottom" as well as derivative thereof (e.g., "horizontally," "downwardly," "upwardly," etc.) should be construed to refer to the orientation as then described or as shown in the drawing under discussion. These relative terms are for convenience of description only and do not require that the apparatus be constructed or operated in a particular orientation.

As used throughout, any ranges disclosed herein are used as shorthand for describing each and every value that is within the range.

<FIG> is a high level schematic diagram flow chart describing the functional aspects of an agricultural sampling system <NUM> according to the present disclosure. The system includes multiple sub-systems which operate in concert and sequence. The sub-systems disclosed herein collectively provide complete processing and chemical analysis of soil samples from collection in the agricultural field, sample preparation and processing, and final chemical analysis. The agricultural material sampled may be soil in one embodiment; however, other types of agricultural materials may be processed and analyzed in the same system including without limitation vegetation/plants, crop residues, forage, manure, feed, milk, and other agricultural related materials of interest in the agricultural, livestock, diary or similar arts. In the context of soil sampling for example which is important to crop production and yield, the agricultural sampling system <NUM> advantageously allows multiple samples to be processed and chemically analyzed simultaneously for different various plant-available nutrients or other parameters such as for example without limitation pH, BpH (buffer pH), etc.. This information may be used to generate nutrient/parameter maps for the agricultural field to determine the appropriate quantities of soil amendments needed in different regions of the field to maximize overall crop production.

In one embodiment, portions of the agricultural sampling system <NUM> may be incorporated onboard a motorized sampling vehicle configured to traverse an agricultural field for collecting and processing soil samples from various zones of the field. This allows a comprehensive nutrient and chemical profile of the field to be accurately generated "on-the-fly" in order to quickly and conveniently identify the needed soil amendments and application amounts necessary in real-time for each zone or region of the field based on quantification of the plant-available nutrient and/or chemical properties in the sample.

The soil sampling system <NUM> generally includes a sample probe collection sub-system <NUM>, a sample preparation sub-system <NUM>, and a chemical analysis sub-system <NUM>. The sample collection sub-system <NUM> and motorized sampling vehicle are fully described in <CIT>. Sample collection sub-system <NUM> generally performs the function of extracting and collecting soil samples from the field. The samples may be in the form of soil plugs or cores. The collected cores are transferred to a holding chamber or vessel for further processing by the sample preparation sub-system <NUM>. Other sampling systems are described in <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT> (<CIT>; and <CIT>; and International Application Nos. <CIT>; and PCT Application Nos. <CIT>;<CIT>; <CIT>; and <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>; <CIT>;<CIT>; <CIT>; and <CIT>. The microfluidic manifold chemical analysis substrate <NUM> disclosed herein is usable in conjunction with any of the system and devices disclosed in the foregoing patent documents, and others.

The sample preparation sub-system <NUM> generally performs the functions of receiving the soil sample cores in a mixer-filter apparatus, volumetric/mass quantification of the soil sample, adding a predetermined quantity or volume of filtered water based on the volume/mass of soil, and mixing the soil and water mixture to produce a soil sample slurry, removing or transferring the slurry from mixer-filter apparatus, and self-cleaning the mixer-filter apparatus for processing the next available soil sample. In some embodiments, the filter may be separate from the mixer.

The chemical analysis sub-system <NUM> generally performs the functions of receiving the soil slurry from a mixer-filter apparatus of sub-system <NUM>, adding extractant, mixing the extractant and slurry in a first chamber to pull out the analytes of interest (e.g. plant available nutrients), centrifuging the extractant-slurry mixture to produce a clear liquid or supernatant, removing or transferring the supernatant to a second chamber, injecting a reagent, holding the supernatant-reagent mixture for a period of hold time to allow complete chemical reaction with reagent, measure the absorbance such as via colorimetric analysis, and assist with cleaning the chemical analysis equipment. In some embodiments, the chemical analysis sub-system <NUM> may be embodied in a microfluidic device or apparatus, as further described herein.

The process described below and in the flow diagrams (see, e.g., <FIG>) may be automatically controlled and executed by the programmable system controller <NUM>. The controller may be part of a controller processing system such as that further described herein and shown in <FIG>, or as disclosed in copending <CIT>. The controller <NUM> is operably coupled to the components of the chemical analysis sub-system <NUM> disclosed herein (e.g., pumps, valves, centrifuge, compressor (air supply), etc.) for controlling the process sequence and flow of fluids (e.g., water, air, slurry, extractant, reagent, supernatant, etc.) through the system to fully process and analyze the soil or other type agricultural sample. <FIG> depicts one embodiment of a programmable system controller <NUM> applicable to the present application.

In some embodiments, the liquid portion may be separated from the soil sample slurry and extractant mixture to produce clear supernatant for chemical analysis using a centrifuge or suitable filter media such an ultrafine microporous filter in lieu of the centrifuge. Suitable centrifuges include centrifuge <NUM> and centrifuge tubes described in commonly-owned <CIT>. A microporous filter <NUM> instead for producing the supernatant include ones such as those described in commonly-owned International Application No. <CIT> and <CIT>. In some embodiments, a microporous sintered metal filter media of suitable shape and structure may be used for the microporous filter. Preferably, the filter media material and shape selected are suitable for backwashing. The microporous filter media selected is configured to produce a clear supernatant suitable for chemical analysis from the slurry and extractant mixture which is suitable for chemical in the microfluidic manifold chemical analysis substrate <NUM> further described herein.

The agricultural sampling system, sub-systems, and related processes/methods disclosed herein may be used for processing and testing soil, vegetation/plants, manure, feed, milk, or other agricultural materials for related parameters of interest. Particularly, embodiments of the chemical analysis portion of the system (chemical analysis sub-system <NUM>) disclosed herein can be used to test for multitude of chemical-related parameters and analytes (e.g. nutrients/chemicals of interest) in other areas beyond soil and plant/vegetation sampling. Some non-limiting examples (including soil and plants) are as follows.

<FIG> is a schematic system diagram showing the control or processing system <NUM> including programmable processor-based central processing unit (CPU) or system controller <NUM> as referenced to herein. System controller <NUM> may include one or more processors, non-transitory tangible computer readable medium, programmable input/output peripherals, and all other necessary electronic appurtenances normally associated with a fully functional processor-based controller. Control system <NUM>, including controller <NUM>, is operably and communicably linked to the different soil sample processing and analysis systems and devices described elsewhere herein via suitable communication links to control operation of those systems and device in a fully integrated and sequenced manner.

Referring to <FIG>, the control system <NUM> including programmable controller <NUM> may be mounted on a stationary support in any location or conversely on a translatable self-propelled or pulled machine (e.g., vehicle, tractor, combine harvester, etc.) which may include an agricultural implement (e.g., planter, cultivator, plough, sprayer, spreader, irrigation implement, etc.) in accordance with one embodiment. In one example, the machine performs operations of a tractor or vehicle that is coupled to an implement for agricultural operations. In other embodiments, the controller may be part of a stationary station or facility.

Control system <NUM>, whether onboard or off-board a translatable machine, generally includes the controller <NUM>, non-transitory tangible computer or machine accessible and readable medium such as memory <NUM>, and a network interface <NUM>. Computer or machine accessible and readable medium may include any suitable volatile memory and non-volatile memory or devices operably and communicably coupled to the processor(s). Any suitable combination and types of volatile or non-volatile memory may be used including as examples, without limitation, random access memory (RAM) and various types thereof, read-only memory (ROM) and various types thereof, hard disks, solid-state drives, flash memory, or other memory and devices which may be written to and/or read by the processor operably connected to the medium. Both the volatile memory and the non-volatile memory may be used for storing the program instructions or software. In one embodiment, the computer or machine accessible and readable non-transitory medium (e.g., memory <NUM>) contains executable computer program instructions which when executed by the system controller <NUM> cause the system to perform operations or methods of the present disclosure including measuring properties and testing of soil and vegetative samples. While the machine accessible and readable non-transitory medium (e.g., memory <NUM>) is shown in an exemplary embodiment to be a single medium, the term should be taken to include a single medium or multiple media (e.g., a centralized or distributed database, and/or associated caches and servers) that store the one or more sets of control logic or instructions. The term "machine accessible and readable non-transitory medium" shall also be taken to include any medium that is capable of storing, encoding or carrying a set of instructions for execution by the machine and that cause the machine to perform any one or more of the methodologies of the present disclosure. The term "machine accessible and readable non-transitory medium" shall accordingly also be taken to include, but not be limited to, solid-state memories, optical and magnetic media, and carrier wave signals.

Network interface <NUM> communicates with the agricultural (e.g. soil or other) sample processing and analysis systems (and their associated devices) described elsewhere (collectively designated <NUM> in <FIG>), and other systems or devices which may include without limitation implement <NUM> having its own controllers and devices. The sample and analysis systems <NUM> therefore specifically include devices such as but are not limited to the chemical analysis substrate <NUM> and process performed by this device which may be sequenced and controlled by controller <NUM>.

The programmable controller <NUM> may include one or more microprocessors, processors, a system on a chip (integrated circuit), one or more microcontrollers, or combinations thereof. The processing system includes processing logic <NUM> for executing software instructions of one or more programs and a communication module or unit <NUM> (e.g., transmitter, transceiver) for transmitting and receiving communications from network interface <NUM> and/or agricultural sample processing and analysis system <NUM> which includes sample preparation sub-system <NUM> and the components described herein further including the closed slurry recirculation flow loop <NUM> components. The communication unit <NUM> may be integrated with the control system <NUM> (e.g. controller <NUM>) or separate from the programmable processing system.

Programmable processing logic <NUM> of the control system <NUM> which directs the operation of system controller <NUM> including one or more processors may process the communications received from the communication unit <NUM> or network interface <NUM> including agricultural data (e.g., test data, testing results, GPS data, liquid application data, flow rates, etc.), and soil sample processing and analysis systems <NUM> generated data. The memory <NUM> of control system <NUM> is configured for preprogrammed variable or setpoint/baseline values, storing collected data, and computer instructions or programs for execution (e.g. software <NUM>) used to control operation of the controller <NUM>. The memory <NUM> can store, for example, software components such as testing software for analysis of soil and vegetation samples for performing operations of the present disclosure, or any other software application or module, images2808 (e.g., captured images of crops), alerts, maps, etc. The system <NUM> can also include an audio input/output subsystem (not shown) which may include a microphone and a speaker for, for example, receiving and sending voice commands or for user authentication or authorization (e.g., biometrics).

The system controller <NUM> communicates bi-directionally with memory <NUM> via communication link <NUM>, network interface <NUM> via communication link <NUM>, display device <NUM> and optionally a second display device <NUM> via communication links <NUM>, <NUM>, and I/O ports <NUM> via communication links <NUM>. System controller <NUM> may further communicate with the soil sample processing and analysis systems <NUM> via wired/wireless communication links <NUM> either via the network interface <NUM> and/or directly as shown.

Display devices <NUM> and <NUM> can provide visual user interfaces for a user or operator. The display devices may include display controllers. In one embodiment, the display device <NUM> is a portable tablet device or computing device with a touchscreen that displays data (e.g., test results of soil, test results of vegetation, liquid application data, captured images, localized view map layer, high definition field maps of as-applied liquid application data, as-planted or as-harvested data or other agricultural variables or parameters, yield maps, alerts, etc.) and data generated by an agricultural data analysis software application and receives input from the user or operator for an exploded view of a region of a field, monitoring and controlling field operations. The operations may include configuration of the machine or implement, reporting of data, control of the machine or implement including sensors and controllers, and storage of the data generated. The display device <NUM> may be a display (e.g., display provided by an original equipment manufacturer (OEM)) that displays images and data for a localized view map layer, as-applied liquid application data, as-planted or as-harvested data, yield data, controlling a machine (e.g., planter, tractor, combine, sprayer, etc.), steering the machine, and monitoring the machine or an implement (e.g., planter, combine, sprayer, etc.) that is connected to the machine with sensors and controllers located on the machine or implement.

<FIG> show various aspects of an embodiment of a microfluidic manifold comprising a polygonal shaped microfluidic manifold chemical analysis substrate <NUM> configured for chemically analyzing the agricultural slurry sample separately processed to produce a supernatant by any suitable means, including for example without limitation by the slurry processing substrate <NUM> disclosed in commonly-owned <CIT> and <CIT>; or the centrifuge <NUM> described in commonly-owned <CIT>. The supernatant separation apparatuses are represented schematically in the block diagram of <FIG>.

Analysis substrate <NUM> is multi-layer structure which comprises a plurality of layers <NUM> which may be permanently bonded together via adhesives, thermal/heat bonding, or other fabrication techniques previously described herein.

In the present embodiment, the functions of extracting the analyte from the agricultural slurry and then subsequently analyzing the centrifugated or filtered slurry (e.g., supernatant) for the concentration of the analyte of interest or other chemical property of the slurry (e.g., pH or BpH) are separated into two discrete and dedicated microfluidic manifold devices. <FIG> is a system block diagram of the overall agricultural sample processing and analysis system. For example, the extractant and slurry mixing function to prepare the sample fluid for chemical analysis by producing the supernatant may be performed by microfluidic manifold slurry processing substrate <NUM> alone. The reagent and supernatant mixing function as well as the analysis and measurement of analyte in the sample fluid may be performed by the chemical analysis substrate <NUM>. Substrate <NUM> includes the reagent/supernatant mixture measurement device such as the flow/slurry analysis cell <NUM> which may be formed by optical absorbance measurement device <NUM> in one embodiment. Device <NUM> is configured to measure and quantify the concentration (e.g. ppm - parts per million) of analyte in the sample fluid. The analysis substrate <NUM> may receive the supernatant from either centrifuge <NUM> or ultrafine microporous filter <NUM> fluidly coupled between the substrates <NUM> and <NUM>. In the latter case, the supernatant may be considered a filtrate from microporous filter <NUM>.

The term "sample fluid" as used herein shall be construed to broadly connote a fluid derived from the agricultural sample such as the reagent-supernatant mixture, extractant-slurry mixture, raw slurry formed of the agricultural material and a carrier such as water, etc..

The chemical analysis substrate <NUM> may generally include the same flow control devices and flow conduits such as the internal pneumatically-actuated microfluidic devices previously described for slurry processing substrate <NUM> in commonly-owned <CIT>.

This includes plural diaphragm-operated micropumps <NUM> and microvalves <NUM>, a chemical analysis cell or device <NUM>, and a network of branched microchannels <NUM> fluidly coupling the devices together. <FIG> shows one non-limiting embodiment and arrangement of fluid interconnections between the microfluidic devices.

Similarly to slurry processing substrate <NUM>, the chemical analysis substrate <NUM> in one non-limiting embodiment may have a five-layer sandwiched construction recognizing that more of less layers may be provided in other embodiments as needed depending on the type of agricultural slurry processing intended to be performed. In order from the planar outer first major surface or side <NUM> to opposite planar outer second major surface or side <NUM>, the adjacent layers of the packaged analysis substrate <NUM> include first outer layer <NUM>, liquid layer <NUM> thereon, air layer <NUM> thereon, fluid distribution layer <NUM> (e.g., air and liquid including but not limited to supernatant, reagent, mixtures thereof, pressurized cleaning/flushing water, etc.) thereon, and second outer layer <NUM> thereon. Outer layer <NUM> defines first major surface or side <NUM> while opposite outer layer <NUM> defines second major surface or side <NUM>. The remaining layers of substrate <NUM> are inner layers. The substrate further includes top side <NUM>, opposite bottom side <NUM>, and pair of opposed lateral sides <NUM>. Major surfaces or sides <NUM>, <NUM> have a greater surface area than other sides of the microfluidic manifold chemical analysis substrate <NUM>. It bears noting that the foregoing layer and side designation use the same reference numbers as slurry processing substrate <NUM> except <NUM> series numbers are assigned in lieu of the original <NUM> series numbering. This shows the correlation between the parts of the two substrates but slightly different configuration and layout of each layer and its constituent components due to their different functions.

Outer layer <NUM> includes a plurality of quick-connect liquid fittings <NUM> and quick connect air valves <NUM> similarly to slurry processing substrate <NUM>. Fluid distribution layer <NUM> is adjacent outer layer <NUM> and includes a plurality of both fluidly separate and/or interconnected microchannels <NUM> for transferring the air and liquids from their applicable sources via fittings <NUM>, <NUM> to and in turn the microfluidic devices (e.g., microvalves <NUM> and micropumps <NUM>) in the microfluidic manifold chemical analysis substrate <NUM>. Each micropump <NUM> and microvalve <NUM> in certain non-limiting embodiments comprises an individual thin and resiliently deformable elastomeric diaphragm <NUM> having an elastic memory. The underside of liquid layer <NUM> comprises a plurality of microchannels <NUM> which fluidly couple the microvalves <NUM> and micropumps <NUM> together. <FIG> shows the fluid interconnections between these microfluidic devices/components formed by the microchannels <NUM>.

In one non-limiting embodiment, the devices and microchannels of the chemical analysis substrate <NUM> may be configured for mixing one or more indicators or reagents (collectively referred to as reagents herein) with the supernatant (filtrated) to create a chemical reaction which changes the color or turbidity of the reagent-supernatant mixture. The reagent operates to change the optical absorbance of light at one or more specified wavelengths.

The absorbance measurement device <NUM> configured to measure and quantify the concentration of the analyte (e.g., soil nutrients or other) or other chemical properties such as pH and/or BpH in the agricultural sample fluid.

Microfluidic manifold chemical analysis substrate <NUM> may have a rectangular cuboid configuration in one embodiment as shown; however, other polygonal shapes may be used. Chemical analysis substrate <NUM> may be used in a vertical orientation in one non-limiting preferred embodiment shown in <FIG> to obtain the benefit gravity assisted agricultural sample fluid flow through the substrate from top to bottom. Other vertical orientations of the rectangular cuboid substrate <NUM> (e.g., long sides extending horizontal instead of vertically seen in FIG. <NUM>), horizontal orientations, or angled orientations with respect to vertical and horizontal may be used in other implementations.

The materials used to construct the individual main layers of chemical analysis substrate <NUM> may include a combination of rigid thermoplastics with flexible elastomeric materials used for the deformable diaphragms associated with each of the micropumps <NUM> and microvalves <NUM>. Transparent polymeric materials may be used in one embodiment to permit visual observation of the fluids being processed in the chemical analysis substrate <NUM>. The rigid plastics may be used to form the overall rigid substrate or body of chemical analysis substrate <NUM> which defines its exposed exterior surfaces and includes an interior patterned to create a plurality of internal microchannels <NUM> and chambers for creating the active microfluidic flow control devices (e.g. diaphragm-operated pumps, valves, mixing chambers, etc.). Examples of thermoplastics (polymers) which may be used include for example without limitation PMMA (polymethyl methacrylate commonly known as acrylic), PC (polycarbonate), PS (polystyrene), and others.

Examples of suitable elastomeric materials which may be used for the diaphragms of the microvalves and micropumps include for example without limitation silicone, PDMS (polydimethylsiloxane), fluorosilicone, neoprene, and others. The pressurized air used to hold the microfluidic valves/pumps closed will permeate through elastomeric diaphragms over time, causing bubbles to develop in the liquid side of the device. These bubbles negatively affect the ability to volumize liquids properly, as the air bubbles displace the otherwise precise fluid volumes that are being manipulated. Fluorosilicone is one preferred non-limiting material due its low gas permeability property which aids in decreasing gas diffusion through the diaphragm over time to combat the foregoing problem.

Referring to <FIG>, the liquid layer <NUM> is shown indicating the flow path of the agricultural sample fluid (e.g., supernatant/filtrate, reagent(s), and reagent-supernatant/filtrate mixture or solution. Bolded flow arrows show the different fluid/reagent inlets and flowpath of the sample liquid through the absorbance measurement device (discussed in further detail below). In this present example for convenience of description, it will be assumed that a microporous filter <NUM> or centrifuge <NUM> is used to produce a clear filtrate or supernatant suitable for chemical analysis by chemical analysis substrate <NUM>. In either case, a clear liquid is produced containing the analyte so that the terms supernatant and filtrate may be used interchangeably herein to refer to the same clear liquid for chemical analysis and analyte quantification.

With continuing reference to <FIG>, the micropumps <NUM>, microvalves <NUM>, and microchannels <NUM> which fluidly coupling these microfluidic devices together are shown in one non-limiting example of numerous possible arrangements. The agricultural sample fluid derived from the agricultural sample progressively flows downwards from top to bottom of the substrate <NUM> to take advantage of gravity assisted flow working in unison with the pumped flow driven by the micropumps. The supernatant/filtrate enters the absorbance analysis substrate <NUM> in liquid layer <NUM> at top via supernatant/filtrate inlet microvalve <NUM>-<NUM> from the microporous filter <NUM>. The supernatant/filtrate flows into a first micropump <NUM>-<NUM> where it is mixed with a first reagent (Reagent <NUM> in figure) flowing from reagent microvalve <NUM>-<NUM>. The supernatant/filtrate and first reagent mixture is pumped by first micropump <NUM>-<NUM> to a second micropump <NUM>-<NUM> via one of several inter-pump microvalves <NUM>-<NUM> as shown which controls flow (i.e. on/off) between the micropumps (only one inter-pump microvalve being labeled in <FIG> for brevity). The supernatant/filtrate and first reagent mixture is pumped by second micropump <NUM>-<NUM> to a third micropump <NUM>-<NUM>, from which is pumped to a fourth micropump <NUM>-<NUM>. As the supernatant/filtrate and first reagent mixture flows through another inter-pump microvalve <NUM>-<NUM> between micropumps <NUM>-<NUM> and <NUM>-<NUM>, it is mixed with a second reagent (Reagent <NUM> in figure) from reagent microvalve <NUM>-<NUM> as shown. Micropump <NUM>-<NUM> then pumps the mixture of the supernatant/filtrate and first-second reagents to a fifth micropump <NUM>-<NUM>. Micropump <NUM>-<NUM> is the final or last micropump located immediately upstream and proximate to the absorbance measurement device <NUM> and measurement passageway <NUM> (further described below). Another inter-pump microvalve <NUM>-<NUM> separates and controls flow between the first micropump <NUM>-<NUM> and measurement device <NUM> as shown. Micropump <NUM>-<NUM> then pumps the supernatant/filtrate and first-second reagent mixture to the fluid inlet <NUM> of the measurement device, the mixture flows through the measurement passageway <NUM> for measurement/quantification of the analyte of interest in the mixture, and exits the device via the fluid outlet <NUM>. In a preferred embodiment, it is highly desirable to have the flow path of the liquid mixture through the measurement passageway <NUM> (flow cell) to be in a vertically upwards direction so that both flow and gravity work together to motivate any entrained air or gas bubbles to rise up and out of the absorbance measurement light path (see, e.g. <FIG> showing vertical orientation of passageway <NUM> represented by dashed lines). The presence of entrained air or gas bubbles in the liquid under optical absorbance analysis in measurement device <NUM> can create errors in measuring the concentration/level of analyte. By using the vertically oriented flowpath and upwards flow of the liquid mixture through the measurement passageway <NUM>, any entrained air/gas bubbles in the measurement passageway rise and are driven out of the line of sight of the optical absorbance measurement device <NUM> to present measurement errors.

In the foregoing example, two reagents are used an introduced into the flow of supernatant/filtrate at two different and physically spaced apart locations in the flow path. This allows time for the first reagent (Reagent <NUM>) to mix with the supernatant/filtrate via pumping through three micropumps <NUM>-<NUM>, <NUM>-<NUM>, and <NUM>-<NUM> prior to adding the second reagent (Reagent <NUM>) in order for the first chemical reaction to be completed and accomplish its intended purpose before introduction of the second reagent and completion of the second reaction followed by subsequent analysis of the mixture in the absorbance measurement device <NUM>.

The optical absorbance measurement device <NUM> will now be further described with reference to the figures and particularly <FIG>. In one embodiment, the absorbance measurement device may be onboard and incorporated directly into the chemical analysis substrate <NUM> via mounting aperture <NUM>. Aperture <NUM> is a through opening in one embodiment which may extend completely through the multiple layers of the substrate between major sides <NUM> and <NUM> and is outwardly open from each end to allow portions of the measurement device housing <NUM> to protrude outwards from the substrate. Aperture <NUM> may have any suitable transverse cross-sectional shape based on the shape of the housing of the measurement device.

Measurement device <NUM> generally includes housing <NUM> comprised of detector sub-housing 6052a and transmitter sub-housing 6052b, upper and lower support plates <NUM>, <NUM>, upper window 6053a, and lower window 6053b. Detector sub-housing 6052a supports light detector printed circuit board (PCB) <NUM> removably attached thereto which comprises light detector 6054a. Transmitter sub-housing 6052b supports transmitter PCB <NUM> removably attached thereto which comprises light transmitter detector 6055a in axial alignment with the light detector. Transmitter PCB <NUM> may be located adjacent to the first major surface or side <NUM> and detector PCB <NUM> may be located adjacent to second major surface or side <NUM> on an opposite side of the analysis substrate <NUM>. Light transmitter 6055a is mounted in light passage <NUM> of the transmitter sub-housing. Light detector 6064a is mounted in light passage <NUM> of the detector sub-housing. Light passages <NUM>, <NUM> communicate directly with the upper and lower windows 6053a, 6053b which may be formed of a transparent material such as glass or polymer operable to transmit light therethrough.

Elongated measurement passageway <NUM> defines a flow cell and is formed between the pair of transparent windows 6053a, 6053b for flowing the agricultural sample fluid (e.g., reagent-supernatant mixture) therethrough for detecting and measuring the analyte absorbance equated to the concentration (e.g., ppm) of analyte present in the fluid. The passageway may be formed in one embodiment by spacer <NUM> disposed between the upper and lower support plates <NUM>, <NUM>. A fluid inlet <NUM> conveys the sample fluid from one of the microchannels <NUM> into the measurement passageway <NUM>. Fluid outlet <NUM> receives and conveys the sample fluid from the measurement passageway <NUM> back into one of the microchannels <NUM>. The fluid inlet and outlet may be at opposite ends of the measurement passageway to extend the flow time of the fluid therethrough. The light transmitter 6055a is arranged to transmit the light through the windows and measurement passageway to the detector 6054a (best shown in <FIG> - note dashed light arrows and solid fluid flow arrows).

Detector sub-housing 6052a and transmitter sub-housing 6052b may be fluidly sealed to the upper and lower windows 6053a, 6053b by seal rings <NUM> which may be an elastomeric O-ring in some embodiments. Seal rings <NUM> (e.g., O-rings) seal the fluid inlet and outlets <NUM>, <NUM> to upper and lower support plates <NUM>, <NUM>.

In one embodiment, the optical absorbance measurement device <NUM> may be detachably mounted to analysis substrate <NUM> by threaded fasteners <NUM>. Fasteners <NUM> may pass through the upper and lower support plates <NUM>, <NUM> and threadably engage one of the layers (e.g., liquid layer <NUM> in the illustrated embodiment). The fasteners further serve to compress the upper and lower support plates and upper and lower together forming the sandwiched structure shown in <FIG>.

The transmitter PCB <NUM> is configured with the circuitry and electronic devices necessary to generate and transmit the light beam through the sample fluid. Similarly, detector PCB <NUM> is configured with the circuitry and electronic devices necessary to receive and process the detected or incident light beam on detector 6054a after passing through the sample fluid in measurement passageway <NUM> to generate an absorbance value associated with the analyte being measured (e.g., soil nutrient or other). Absorbance is a dimensionless value and often referred to as simply an absorbance value or unit. Each PCB <NUM>, <NUM> has an associated electronics cable connector <NUM> which provides operable coupling of electric power to the boards for the devices therein including the transmitter and detector, and two-way communication links between the system controller <NUM> and board electronics (see, e.g. <FIG>).

Optical absorbance measurement device <NUM> operates in a known manner similar to commercially-available absorbance measurement or spectrophotometric devices. Measurement device <NUM> is configured and operable to measure the absorbance of light passing through the agricultural sample fluid which can be equated to concentration of the analyte of interest (e.g., ppm).

The chemical reaction that takes place to create absorbance in the agricultural sample fluid (e.g., reagent-supernatant mixture or solution) can vary with temperature. For example, these chemical reactions occur faster at higher fluid temperatures and slower at lower fluid temperatures. Accordingly, an absorbance measurement taken by optical absorbance measurement device <NUM> at one temperature when equated to a specific concentration of analyte in the sample fluid might not accurately reflect the actual concentration.

According to one aspect of the present disclosure, an automated analysis system comprising programmable controller <NUM> operating in conjunction with optical absorbance measurement device <NUM> and temperature sensor <NUM> is provided which is configured to generate a temperature-compensated concentration of analyte in the agricultural sample fluid. Advantageously, this results in more accurate determination of the actual concentration of the analyte (e.g., soil nutrient or other chemical) present in the sample fluid than an absorbance measurement obtained without regard for the temperature of the fluid.

Temperature sensor <NUM> may be any suitable commercially-available temperature sensor such as a thermistor or thermocouple. Temperature sensor <NUM> may be embedded internally in the substrate proximate to but not necessarily contacting the sample liquid. Accordingly, sensor <NUM> may be preferably located upstream of absorbance measurement device <NUM> and disposed proximate to in any of the microchannels <NUM>, micropumps <NUM>, or microvalves <NUM>. To accurately measure the temperature of the agricultural sample fluid (e.g., reagent-supernatant solution), sensor <NUM> is preferably upstream of absorbance measurement device in close proximity thereto. In one non-limiting embodiment, temperature sensor <NUM> may be disposed proximate to the final micropump <NUM> upstream of measurement device <NUM> to measure the temperature of the agricultural sample fluid in the pump. Sensor <NUM> may be separated from the pump chamber <NUM> by a partition wall <NUM> formed by a portion of the substrate (e.g., liquid layer <NUM> in one embodiment as best shown in <FIG>). In other possible embodiments, the temperature sensor may be located proximate to one of the microchannels <NUM> between the final micropump <NUM> and measurement device <NUM>, or the microvalve <NUM> between the final micropump and measurement device. In some embodiments, the temperature sensor <NUM> may be in physical and direct contact with the agricultural sample fluid rather than separated therefrom by a partition wall.

Referring to <FIG>, temperature sensor <NUM> is mounted in a measurement bore <NUM> formed and extending through the layers of the analysis substrate <NUM> to the final micropump <NUM>, a microchannel <NUM>, or microvalve <NUM>. In one embodiment, bore <NUM> may have a cylindrical configuration with round cross-sectional shape and completely penetrates one of the outer layers <NUM> or <NUM>. In one embodiment, bore <NUM> may penetrate outer layer <NUM> as shown. Temperature sensor <NUM> is disposed at the terminal closed end <NUM> of measurement bore <NUM> adjacent to partition wall <NUM>.

Measurement bore <NUM> permits the wire leads <NUM> of temperature sensor <NUM> to be routed from the sensor through the substrate layers to system controller <NUM> as shown in the system block diagram of <FIG>. Sensor <NUM> is configured to measure a real-time temperature of the agricultural sample fluid and transmit the measure real-time temperature to the controller. As shown, the optical absorbance measurement device <NUM> is also operably coupled to system controller <NUM> for transmitting the measured absorbance of light thereto which is indicative of the concentration of analyte present in the sample fluid. The controller uses the real-time temperature and absorbance value to execute a software routine which generates a temperature-compensated concentration of analyte.

To determine the temperature-compensated concentration of analyte, the controller may use and is preprogrammed with a set of curves similar to those shown in the graphs of <FIG> and <FIG>. <FIG> is a graph of a base calibration curve showing absorbance units on one scale versus concentration of a selected analyte (ppm) on the other scale (e.g., phosphorous, nitrogen, potassium, etc.). This curve is used by controller <NUM> to correlate a measured absorbance obtained by optical absorbance measurement device <NUM> to a concentration of the selected analyte in the agricultural sample fluid. The graph is constructed by conducting multiple test runs to measure the absorbance of a plurality of different calibration standard fluids (or simply "standards") each having a different and unique known concentration (ppm) of the selected test analyte of interest.

<FIG> is a graph showing absorbance units on one scale versus temperature on the other scale. Shown is one example of a temperature compensation curve developed empirically by testing a first calibration standard fluid having a known concentration (ppm) of the selected analyte (e.g., <NUM> ppm of phosphorous in the example graph) over a range of different temperatures which might be encountered when testing the agricultural sample fluid in measurement device <NUM>. Accordingly, the curve indicates the temperature compensation relationship of the selected analyte for the first calibration standard fluid of known concentration. The temperature compensation curve shows the shift in absorbance values/units as the sample fluid temperature changes. The temperature compensation curve may be developed by running the first calibration standard fluid through the analysis substrate <NUM> and obtaining measurements from the measurement device <NUM> and temperature sensor <NUM>.

Although only a single curve is shown in <FIG> for simplicity, a plurality of different temperature compensation curves would be generated in a similar manner for each of a multitude of different calibration standard fluids each having a unique and different known concentration of the selected analyte which were originally used to construct the base calibration curve of <FIG>. The base calibration curve may be automatically adjusted by controller <NUM> in real-time based on the real-time temperature measurements from temperature sensor <NUM> and the plural temperature compensation curves indicating the variance of absorbance values/units with temperature for the different calibration standards. Thus, the base calibration curve is adjusted up or down in real-time by controller <NUM> to compensate for the real-time temperature of the agricultural sample fluid measured by temperature sensor <NUM> which is communicated to the controller.

In operation when testing an agricultural sample fluid for the concentration of an analyte in microfluidic manifold analysis substrate <NUM>, optical absorbance measurement device <NUM> measures the absorbance of the sample fluid. The absorbance value is transmitted to system controller <NUM> along with a corresponding real-time temperature measure of the fluid captured by temperature sensor <NUM>. The controller adjusts the base calibration curve as needed based on the measured real-time temperature using graph of <FIG>. Using the temperature-compensated base calibration curve, the controller then used the graph of <FIG> to correlate the measured absorbance of the selected analyte to a corresponding concentration of the analyte (e.g., ppm). Accordingly, the controller is configurable to determine a temperature-compensated concentration of the analyte based on the actual measured absorbance and real-time temperature.

Although the temperature-compensated concentration of analyte generated by controller <NUM> is disclosed above with respect to the optical absorbance measurement device <NUM> and temperature sensor <NUM> used in conjunction with a microfluidic manifold analysis support structure <NUM>, other embodiments contemplated may not use a microfluidic manifold for mixing the reagent and supernatant/filtrate (agricultural sample fluid). The agricultural sample fluid may instead be processed through non-microfluidic devices (e.g., pumps, valves, flow conduits, etc.) fluidly coupled together via tubing/piping and used in conjunction with the measurement device <NUM>, temperature sensor <NUM>, and controller <NUM> disclosed to obtain the same functionality and temperature-compensated analyte concentration results albeit in a less compact form factor. Preferably in such conventional flow systems, the temperature of the sample fluid should preferably still be measured upstream of the measurement device proximate to the inlet of the device for accurate temperature and analyte measurements. Examples of non-microfluidic slurry and agricultural sample fluid systems and devices which may be used include those disclosed in commonly-owned International Patent Applications <CIT> and <CIT>.

The following are non-limiting examples.

Example <NUM> - A microfluidic manifold for analyzing an agricultural sample comprising: a substrate comprising a plurality of microfluidic devices fluidly coupled together by microchannels configured to convey a sample fluid derived from the agricultural sample; a measurement device mounted to the substrate, the measurement device configured to measure an absorbance value associated with an analyte in the sample fluid; a temperature sensor configured to measure a real-time temperature of the sample fluid; and a programmable controller operably coupled to the measurement device and temperature sensor, the controller being configured to determine a temperature-compensated concentration of the analyte based on the measured absorbance value and real-time temperature.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the temperature sensor is embedded internally in the substrate proximate to the sample fluid.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the temperature sensor is mounted in a measurement bore formed through the substrate from a first outer surface of the substrate.

Example <NUM> - the microfluidic manifold according to <NUM>, wherein the microfluidic device includes at least one micropump, the temperature sensor being configured to measure the real-time temperature of the sample fluid in the at least one micropump.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the measurement bore comprises a closed terminal end separated from the micropump by a partition wall formed from the substrate.

Example <NUM> - the microfluidic manifold according to Examples <NUM> or <NUM>, wherein the at least one micropump is located upstream of and proximate to the measurement device.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the at least one micropump is fluidly coupled to the measurement device through a microvalve configured to control flow of the sample fluid between the at least one micropump and the measurement device.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the measurement bore and temperature sensor are configured and arranged to measure the real-time temperature of the sample fluid upstream of the measurement device.

Example <NUM> - the microfluidic manifold according to any one of Examples <NUM>-<NUM>, wherein the temperature sensor is a thermistor or a thermocouple.

Example <NUM> - the microfluidic manifold according to any one of Examples <NUM>-<NUM>, wherein the controller is configured to receive the absorbance value from the measurement device and correlate the absorbance value to the concentration of analyte using a preprogrammed base calibration curve.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the controller is configured to: receive the real-time temperature of the sample fluid from the temperature sensor; and automatically adjust the base calibration curve based on the real-time temperature.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the controller uses a plurality of preprogrammed temperature compensation curves to adjust the base calibration curve based on the real-time temperature of the sample fluid received from the temperature sensor.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the temperature compensation curves provide a variance of absorbance versus a range of temperatures for plural calibration standard fluids each having a different know concentration of the analyte.

Example <NUM> - the microfluidic manifold according to any one of Examples <NUM>-<NUM>, wherein the measurement device is an optical absorbance measurement device.

Example <NUM> - the microfluidic manifold according to any one of Examples <NUM>-<NUM>, wherein the measurement device is mounted in a mounting aperture extending through and between opposite first and second major sides of the substrate.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the measurement device includes a transmitter printed circuit board mounted adjacent to the first major side and an opposing detector printed circuit board mounted adjacent to the second major side.

Example <NUM> - the microfluidic manifold according to any one of Examples <NUM>-<NUM>, wherein the measurement device includes an elongated measurement passageway formed between a pair of transparent windows, an inlet fluidly coupled to the measurement passageway and configured to receive the sample fluid from one of the microchannels, and an outlet fluidly coupled to the measurement passageway and configured to receive the sample fluid therefrom and return the sample fluid to another one of the microchannels.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the measurement passageway is vertically oriented and operable to carry entrained gas in the sample fluid out of the measurement passageway.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the sample fluid flows upwards in the measurement passageway.

Example <NUM> - the microfluidic manifold according to any one of Examples <NUM>-<NUM>, wherein the measurement device is threadably coupled to the substrate by fasteners.

Example <NUM> - the microfluidic manifold according to any one of Examples <NUM>-<NUM>, wherein the substrate is multi-layered comprised of a plurality of layers formed of polymeric material joined together.

Example <NUM> - the microfluidic manifold according to Example <NUM>, wherein the microfluidic devices are embedded within the layers between opposing outer major surfaces of the substrate.

Example <NUM> - A method for analyzing an agricultural sample comprising: providing a substrate comprising a plurality of microfluidic devices fluidly coupled together by microchannels configured to convey a sample fluid derived from the agricultural sample; measuring an absorbance value associated with an analyte in the sample fluid with a measurement device; measuring a real-time temperature of the sample fluid with a temperature sensor; and determining with programmable controller a temperature-compensated concentration of the analyte based on the measured absorbance value and real-time temperature.

Example <NUM> - the method according to Example <NUM>, wherein the controller receives the absorbance value from the measurement device and correlates the absorbance value to the concentration of analyte using a preprogrammed base calibration curve.

Example <NUM> - the method according to Example <NUM>, wherein the controller receives the real-time temperature of the sample fluid from the temperature sensor and automatically adjusts the base calibration curve based on the real-time temperature.

Example <NUM> - the method according to Example <NUM>, wherein the controller uses a plurality of preprogrammed temperature compensation curves to adjust the base calibration curve based on the real-time temperature of the sample fluid received from the temperature sensor.

Example <NUM> - the method according to Example <NUM>, wherein the temperature compensation curves provide a variance of absorbance versus a range of temperatures for different calibration standard fluids each having a different know concentration of the analyte.

Example <NUM> - the method according to any one of Examples <NUM>-<NUM>, wherein the measurement device is an optical absorbance measurement device.

Example <NUM> - the method according to any one of Examples <NUM>-<NUM>, wherein the step of measuring the absorbance value associated with the analyte further comprises flowing the sample fluid vertically upwards through a measurement passageway of the measurement device.

Claim 1:
A microfluidic manifold for analyzing an agricultural sample comprising:
a substrate (<NUM>) comprising a plurality of microfluidic devices fluidly coupled together by microchannels (<NUM>) configured to convey a sample fluid derived from the agricultural sample;
a measurement device (<NUM>) mounted to the substrate (<NUM>), the measurement device (<NUM>) configured to measure an absorbance value associated with an analyte in the sample fluid;
a temperature sensor (<NUM>) configured to measure a real-time temperature of the sample fluid; and
a programmable controller (<NUM>) operably coupled to the measurement device (<NUM>) and temperature sensor (<NUM>),
characterized in that
the programmable controller (<NUM>) is configured to determine a temperature-compensated concentration of the analyte based on the measured absorbance value and real-time temperature.