Integrated device, system, and method for pathogen detection in water

Abstract

The present disclosure provides autonomous devices, systems incorporating the devices, and methods of using the devices for carrying out assays, such as chemical and / or biological assays. The systems include a sample board including a substrate, multiple fluorescence emissions sensors arranged in a grid on the substrate, and multiple fluorescence excitation sources each positioned adjacent to a sensor. A sample tray include multiple reaction wells arranged in a grid. A computing device causes the fluorescence excitation sources to illuminate the reaction wells when the sample board is positioned on the sample tray. Each reaction well includes an aqueous medium, which may contain, for example, a target bacteria. The computing device detects a fluorescence signal from each reaction well with a corresponding sensor, and determines a water quality metric based on the fluorescence signals. Other embodiments are described and claimed.

Description

INTEGRATED DEVICE, SYSTEM, AND METHOD FOR PATHOGEN DETECTION IN WATERGOVERNMENT SUPPORT STATEMENT

[0001] This invention was made with government support under Grant No. R / 90-2-20 awarded by the National Oceanic and Atmospheric Administration (NOAA). The government has certain rights in the invention.”CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit under 35 USC § 119(e) of U.S. Provisional Application Serial No. 63 / 539,360, filed on September 20, 2023, the entire disclosure of which is incorporated herein by reference.TECHNICAL FIELD

[0003] The present disclosure relates to devices and methods for water quality analysis. More particularly, the disclosure relates to an integrated water quality monitoring device and methods of using the device.BACKGROUND

[0004] Water is one of the most crucial natural resources for living beings. Water is utilized in daily activities such as cooking, bathing, drinking, and recreation. The Environmental Protection Agency (EP A) provides a list of water pollutant testing guidelines in 40 C.F.R. Part 136. Testing procedures include, for example, biological indicators (bacteria, aquatic toxicity), inorganic indicators (metals), organic indicators, pesticides, radiological indicators, and microbiological indicators (bacteria, protozoa).

[0005] Monitoring coliform bacteria (total coliforms, E. coli and E. faecalis) is used for microbiological ambient water quality assessments and serves as proxy to the presence of other, more pathogenic, fecal bacteria. Tests developed for bacteria detection should consider sensitivity, specificity, robustness, repeatability, cost, and speed.

[0006] Conventional methods for fecal bacteria monitoring in water include cell culture, polymerase chain reaction (PCR), and membrane filtration. These methods have high sensitivity and specificity but require cold transport to a laboratory, expensive equipment, trained personnel, and may require days to produce and communicate results. On the other hand, biosensors are being developed that can detect a pathogens in a portable, automated, in-situ fashion and that can provide information in near real-time.

[0007] Biosensors are devices capable of transducing a biological or chemical signal into a mechanical or electrical signal capable of being measured, calibrated, and displayed. Developing a biosensor requires knowledge from different fields, such as engineering, physics, biology, and chemistry. Successful examples of biosensors can be found in the fields of medicine, in the military, and in environmental monitoring applications. One of the most important areas for the application of biosensors is in health care because of the increasing relevance for early, rapid, and in-si tu diagnostics.BRIEF SUMMARY

[0008] The present disclosure provides devices, systems, and methods for water quality analysis. In some embodiments, a system comprises a sample board, a sample tray, and a computing device coupled to the sample board. The sample board comprises a substrate, a plurality of fluorescence emission sensors coupled to the substrate, wherein the fluorescence emission sensors are arranged in a grid defined by a predetermined pitch, and a plurality of fluorescence excitation sources coupled to the substrate, wherein each of the fluorescence excitation sources is positioned adjacent to a fluorescence emission sensor. The sample tray comprises a plurality of reaction wells arranged in a grid defined by the predetermined pitch, wherein each reaction well comprises a reagent to carry out a reaction, and wherein each reaction well is transparent. The computing device is configured to cause the plurality of fluorescence excitation sources to illuminate the plurality of reaction wells when each reaction well of the sample tray includes an aqueous medium and the sample board is positioned on the sample tray, to detect a fluorescence signal from each of the plurality of reaction wells with a corresponding fluorescence emission sensor, wherein each fluorescence signal is indicative of whether fluorescent activity is detected in the corresponding reaction well, and to determine a water quality metric based on the fluorescence signal from each of the plurality of reaction wells.

[0009] In some embodiments, each fluorescence excitation source comprises an ultraviolet (UV) light-emitting diode (LED); and each fluorescence emission sensor comprises a photodiode.

[0010] In some embodiments, the sample board further comprises a control system comprising a controller and an ammeter coupled to the controller. To detect the fluorescence signal from each of the plurality of reaction wells comprises to sequentially select, by the controller of the sensor board, a photodiode associated with each reaction well, and to measure, by the controller, current from the photodiode using the ammeter in response to selection of the photodiode. In some embodiments, the control system of the sample board further comprises aplurality of multiplexers, wherein each multiplexer comprises an input coupled to an output of a photodiode, a first output coupled to ground, and a second output coupled to the ammeter. To sequentially select the photodiode associated with each reaction well comprises to selectively cause a first multiplexer of the plurality of multiplexers to connect the input of the first multiplexer to the second output of the first multiplexer and to cause remaining multiplexers of the plurality of multiplexers to connect the input of each respective multiplexer to the first output of each respective multiplexer.

[0011] In some embodiments, to determine the water quality metric comprises to determine a number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity; and to determine a most probable number (MPN) metric as a function of the number of the reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity. In some embodiments, to determine the number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity comprises to determine an inflection point for the corresponding fluorescence signal. In some embodiments, to determine the number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity comprises, for each fluorescence signal, to determine a change in fluorescence signal, and to determine whether the change in fluorescence signal has a predetermined relationship with a predetermined threshold. In some embodiments, the fluorescence signal comprises a current received from a photodiode, and to determine whether the change in fluorescence signal has the predetermined relationship with the predetermined threshold comprises to determine whether a change in current is greater than a predetermined change in current. In some embodiments, the threshold change in current comprises 200 nA, 20 nA, or 10 nA.

[0012] In some embodiments, the system further comprises a heater coupled to a controller. The controller is configured to heat the sample tray and the sample board at a predetermined temperature when the sample board is positioned on the sample tray.

[0013] In some embodiments, the computing device is further configured to continue to heat the device, illuminate the plurality of reaction wells, and detect the fluorescence signal from each of the plurality of reaction wells over a first time period between about 3.3 hours to less than 24 hours, and to determine the water quality metric based on the fluorescence signal from each of the plurality of reaction wells after expiration of the first time period.

[0014] In some embodiments, the aqueous medium comprises lake water, river water, or ocean water.

[0015] In some embodiments, the predetermined temperature is between about 20 °C to about 60 °C.

[0016] In some embodiments, the reaction is a biological reaction, a chemical reaction, a biochemical reaction, or any combination thereof.

[0017] In some embodiments, the reagent comprises a nutrient and optionally an inhibitor. In some embodiments, the nutrient comprises o-nitrophenyl-P-D-galactopyranoside (ONPG), 4-methylumbelliferyl-P-D-glucuronide (MUG), 4-methylumbelliferyl-P-D-glucoside, or any combination thereof. In some embodiments, the inhibitor comprises a salt, a sugar, a phenol, or any combination thereof.

[0018] In some embodiments, the sample tray is formed from a transparent, impermeable polymer, wherein the impermeable polymer is thermoplastic, thermoset, machinable, manufacturable, translucent, biocompatible, chemically inert, resistant to ultraviolet light, or any combination thereof. In some embodiments, the impermeable polymer comprises substantially no autofluorescence. In some embodiments, the impermeable polymer comprises methyl methacrylate, polystyrene, polycarbonate, polypropylene, polyethylene, a cycloolefin polymer, and any combination thereof.

[0019] The present disclosure also provides a method for water quality analysis comprising loading an aqueous medium into a plurality of reaction wells of a sample tray, wherein each reaction well comprises a reagent to carry out a reaction, and wherein each reaction well is transparent; sealing the aqueous medium in the plurality of reaction wells; positioning a sample board on the sample tray after sealing the aqueous medium, wherein the sample board comprises a plurality of fluorescence excitation sources and a plurality of fluorescence emission sensors, and wherein when positioned on the sample tray, each fluorescence emission sensor is positioned to sense emissions from a corresponding reaction well; heating the sample tray and the sample board at a predetermined temperature; illuminating each of the plurality of reaction wells of the device with the plurality of fluorescence excitation sources; detecting a fluorescence signal from each of the plurality of reaction wells with a corresponding fluorescence emission sensor of a plurality of fluorescence emission sensors, wherein each fluorescence signal is indicative of whether fluorescent activity is detected in the corresponding reaction well; and determining, by a computing device, a water quality metric based on the fluorescence signal from each of the plurality of reaction wells.

[0020] In some embodiments, each fluorescence excitation source comprises an ultraviolet (UV) light-emitting diode (LED), and each fluorescence emission sensor comprises a photodiode.

[0021] In some embodiments, detecting the fluorescence signal from each of the plurality of reaction wells comprises sequentially selecting, by a controller of the sample board, a photodiode associated with each reaction well; and measuring, by the controller, current from the photodiode using an ammeter in response to selecting the photodiode. In some embodiments, sequentially selecting the photodiode comprises causing a multiplexer coupled to the photodiode to output current from the photodiode to the ammeter and causing a multiplexer coupled to each of the remaining photodiodes to output current to ground.

[0022] In some embodiments, determining the water quality metric comprises determining a number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity; and determining a most probable number (MPN) metric as a function of the number of the reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity. In some embodiments, determining the number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity comprises determining an inflection point for the corresponding fluorescence signal. In some embodiments, determining the number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity comprises, for each fluorescence signal, determining a change in fluorescence signal, and determining whether the change in fluorescence signal has a predetermined relationship with a predetermined threshold. In some embodiments, the fluorescence signal comprises a current received from a photodiode, and wherein determining whether the change in fluorescence signal has the predetermined relationship with the predetermined threshold comprises determining whether a change in current is greater than a predetermined change in current. In some embodiments, the threshold change in current comprises 200 nA, 20 nA, or 10 nA.

[0023] In some embodiments, the method further comprises continuing to heat the device, illuminate the plurality of reaction wells, and detect the fluorescence signal from each of the plurality of reaction wells over a first time period between about 3.3 hours to less than 24 hours; and determining the water quality metric based on the fluorescence signal from each of the plurality of reaction wells after expiration of the first time period.

[0024] In some embodiments, the aqueous medium comprises lake water, river water, or ocean water.

[0025] In some embodiments, the predetermined temperature is between about 20 °C to about 60 °C.

[0026] In some embodiments, the reaction is a biological reaction, a chemical reaction, a biochemical reaction, or any combination thereof.

[0027] In some embodiments, the reagent comprises a nutrient and optionally an inhibitor. In some embodiments, the nutrient comprises o-nitrophenyl-P-D-galactopyranoside (ONPG), 4-methylumbelliferyl-|3-D-glucuronide (MUG), 4-methylumbelliferyl-0-D-glucoside, or any combination thereof. In some embodiments, the inhibitor comprises a salt, a sugar, a phenol, or any combination thereof.

[0028] In some embodiments, the sample tray is formed from a transparent, impermeable polymer, wherein the impermeable polymer is thermoplastic, thermoset, machinable, manufacturable, translucent, biocompatible, chemically inert, resistant to ultraviolet light, or any combination thereof. In some embodiments, the impermeable polymer comprises substantially no autofluorescence. In some embodiments, the impermeable polymer comprises methyl methacrylate, polystyrene, polycarbonate, polypropylene, polyethylene, a cycloolefin polymer, and any combination thereof.

[0029] The present disclosure also provides a device comprises a substrate, a plurality of fluorescence emission sensors coupled to the substrate, a plurality of fluorescence excitation sources coupled to the substrate, and a control system coupled to the fluorescence emission sensors and the fluorescence excitation sources. The fluorescence emission sensors are arranged in a grid defined by a predetermined pitch. Each of the fluorescence excitation sources is positioned adjacent to a fluorescence emission sensor. The control system is configured to activate the fluorescence excitation sources and receive a fluorescence signal from each of the fluorescence emission sensors while the fluorescence excitation sources are activated.

[0030] In some embodiments, each fluorescence excitation source comprises an ultraviolet (UV) light-emitting diode (LED); and each fluorescence emission sensor comprises a photodiode.

[0031] In some embodiments, the control system comprises a control system comprising a controller and an ammeter coupled to the controller. The controller is configured to sequentially select, by the controller of the sensor board, a photodiode from the plurality of photodiodes; and measure, by the controller, current from the photodiode using the ammeter in response to selection of the photodiode. In some embodiments, the control system further comprises a plurality of multiplexers. Each multiplexer comprises an input coupled to an output of a photodiode, a first output coupled to ground, and a second output coupled to the ammeter. To sequentially select the photodiode comprises to selectively cause a first multiplexer of the plurality of multiplexers to connect the input of the first multiplexer to the second output of thefirst multiplexer and to cause remaining multiplexers of the plurality of multiplexers to connect the input of each respective multiplexer to the first output of each respective multiplexer.

[0032] The foregoing has outlined rather broadly the features and technical advantages of the present disclosure in order that the detailed description that follows may be better understood. Additional features and advantages of the disclosure will be described hereinafter that form the subject of the claims of this application. It should be appreciated by those skilled in the art that the conception and the specific embodiments disclosed may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present disclosure. It should also be realized by those skilled in the art that such equivalent embodiments do not depart from the spirit and scope of the disclosure as set forth in the appended claims.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0033] A detailed description of the invention is hereafter described with specific reference being made to the drawings in which:

[0034] FIG. 1 shows a system according to the present disclosure including a sample board and corresponding sample tray;

[0035] FIG. 2 shows a top view of a sample board according to the present disclosure;

[0036] FIG. 3 shows a side view of a sample board positioned on a sample tray according to the present disclosure;

[0037] FIG. 4 depicts a schematic diagram of a control system for a sample board of the system in accordance with some embodiments of the present disclosure;

[0038] FIGS. 5 and 6 are a simplified flow diagram of at least one method for water quality analysis that may be performed by the system of FIGS. 1-4; and

[0039] FIG. 7 depicts illustrative experimental results that may be achieved by a system in accordance with the present disclosure.DETAILED DESCRIPTION

[0040] Various embodiments are described below with reference to the drawings in which like elements generally are referred to by like numerals. The relationship and functioning of the various elements of the embodiments may better be understood by reference to the following detailed description. However, embodiments are not limited to those illustrated in the drawings or explicitly described below.

[0041] Referring now to FIG. 1, an embodiment of a system 100 of the present disclosure is depicted. As shown, the illustrative system 100 includes a computing device 102 coupled to asample board 104 and a controller 110. In use, as described further below, a sample tray 106 may be loaded with a sample of an aqueous medium such as fresh water (lake water, river water) or marine water (salt water, sea water, ocean water) for water quality analysis. The sample board 104 is positioned on the sample tray 106. The sample tray 106 is brought to a predetermined temperature using a heater 108, and the sample board 104 is used to measure fluorescence generated by the sample in the sample tray 106. Based on those measurements, the computing device 102 determines a water quality metric. As described further below, the system 100 may determine the water quality metric in a shorter amount of time (e.g., 12 hours, 6 hours, 3 hours, or another time) as compared to conventional water quality analysis, which may require at least 24 hours of incubation.

[0042] The computing device 102 may be embodied as any type of device capable of performing the functions described herein. For example, the computing device 102 may be embodied as, without limitation, a microcomputer, a workstation, a desktop computer, a laptop computer, a tablet computer, a smartphone, a server, a rack-mounted server, a blade server, a network appliance, a web appliance, a consumer electronic device, a distributed computing system, a multiprocessor system, and / or any other computing device capable of performing the functions described herein. Additionally, in some embodiments, the computing device 102 may be embodied as a “virtual server” formed from multiple computing devices distributed across a network and operating in a public or private cloud. Accordingly, although the computing device 102 is illustrated in FIG. 1 as embodied as a single computing device, it should be appreciated that the computing device 102 may be embodied as multiple devices cooperating together to facilitate the functionality described below. As shown in FIG. 1 , the illustrative computing device 102 includes a processor 120, an TO subsystem 122, memory 124, a data storage device 126, and a communication subsystem 128. Of course, the computing device 102 may include other or additional components, such as those commonly found in a desktop computer (e.g., various input / output devices), in other embodiments. Additionally, in some embodiments, one or more of the illustrative components may be incorporated in, or otherwise form a portion of, another component. For example, the memory 124, or portions thereof, may be incorporated in the processor 120 in some embodiments.

[0043] The processor 120 may be embodied as any type of processor or compute engine capable of performing the functions described herein. For example, the processor may be embodied as a single or multi-core processor(s), digital signal processor, microcontroller, or other processor or processing / controlling circuit. Similarly, the memory 124 may be embodied as any type of volatile or non-volatile memory or data storage capable of performing the functionsdescribed herein. In operation, the memory 124 may store various data and software used during operation of the computing device 102 such as operating systems, applications, programs, libraries, and drivers. The memory 124 is communicatively coupled to the processor 120 via the I / O subsystem 122, which may be embodied as circuitry and / or components to facilitate input / output operations with the processor 120, the memory 124, and other components of the computing device 102. For example, the I / O subsystem 122 may be embodied as, or otherwise include, memory controller hubs, input / output control hubs, firmware devices, communication links (i.e., point-to-point links, bus links, wires, cables, light guides, printed circuit board traces, etc.) and / or other components and subsystems to facilitate the input / output operations. In some embodiments, the I / O subsystem 122 may form a portion of a system-on-a-chip (SoC) and be incorporated, along with the processor 120, the memory 124, and other components of the computing device 102, on a single integrated circuit chip.

[0044] The data storage device 126 may be embodied as any type of device or devices configured for short-term or long-term storage of data such as, for example, memory devices and circuits, memory cards, hard disk drives, solid-state drives, or other data storage devices. The communication subsystem 128 of the computing device 102 may be embodied as any communication circuit, device, or collection thereof, capable of enabling communications between the computing device 102 and other remote devices. The communication subsystem 128 may be configured to use any one or more communication technology (e.g., wireless or wired communications) and associated protocols (e.g., Ethernet, Bluetooth®, Bluetooth Low Energy (BLE), Wi-Fi®, WiMAX, 3G LTE, 5G, etc.) to effect such communication.

[0045] The controller 110 may be illustratively embodied as a microcontroller, such as an Arduino microcontroller, a microcomputer, such as a Raspberry Pi microcomputer, or other control device, is coupled to the heater 108. As described above, the controller 110 ensures that the heater 108 reaches and maintains a predetermined temperature. The heater 108 may be embodied as an incubation heater, a temperature chamber, a hot plate, or a thermoelectric heater, for example. The system 100 may also include one or more thermocouples, thermometers, thermistors, resistance temperature detectors, or other temperature sensors in order to control temperature of the heater 108 and thus the sample tray 106 and / or other components of the system 100.

[0046] The sample tray 106 includes multiple reaction wells 160 arranged in a grid or other array. As described above, each reaction well 160 may be filled with an aqueous medium such as sampled water. Each well 160 is separate, independent, and sealed off from the other wells such that there is no cross-contamination between wells. The reaction well 160 maycomprise any shape and in some embodiments, if there are multiple reaction wells, the shape of each well may be the same or independently selected. For example, the shape of a reaction well 160 may be independently selected from circular, oval, square, rectangular, and triangular. The reaction well 160 may be of any desired dimension and any desired depth / height. For example, if the reaction well 160 has a rectangular or square shape, the length and width of each side may be independently selected from about 1 mm to about 100 mm, such as from about 1 mm to about 80 mm, from about 1 mm to about 60 mm, from about 1 mm to about 40 mm, from about 1 mm to about 20 mm, from about 1 mm to about 10 mm, from about 10 mm to about 100 mm, from about 30 mm to about 100 mm, from about 50 mm to about 100 mm, or from about 70 mm to about 100 mm. Similarly, in some embodiments, the depth / height of the reaction well 160 may be independently selected from about 1 mm to about 10 mm. For example, the height may be about 1 mm to about 9 mm, about 1 mm to about 8 mm, about 1 mm to about 7 mm, about 1 mm to about 6 mm, about 1 mm to about 5 mm, about 1 mm to about 4 mm, about 1 mm to about 3 mm, or about 1 mm to about 2 mm.

[0047] The sample tray 106 and / or the walls of the reaction wells 160 may be formed from a polymer material that is transparent to ultraviolet (UV) and visible light. The polymer may be impermeable to solids, liquids, and / or gasses. The polymer may be a thermoset polymer or a thermoplastic polymer, for example. Additionally, in some embodiments the polymer is transparent, semi-transparent, machinable, manufacturable, translucent, biocompatible, chemically inert, resistant to ultraviolet light, and / or any combination thereof. The polymer comprises little or no autofluorescence. After being filled with the aqueous medium, the reaction wells 160 may be sealed, for example by application of an impermeable top layer.

[0048] In some embodiments, the sample tray 106 may be a standard disposable sample tray used in microbiology labs such a Quanti-Tray 2000® sample tray, commercially available from IDEXX. The Quanti-Tray 2000 is a platform that captures and divides 100 mL of sample water into 96 discrete wells, including 48 large wells and 48 small wells. The illustrative system 100 analyzes the 48 large wells. Accordingly, in the illustrative embodiment, the sample tray 106 may include 48 reaction wells 160, arranged in a regular grid that is eight wells by six wells. Other numbers and / or arrangements of reaction wells 160 may be used in other embodiments. For example, in some embodiments the sample tray 106 may have any number of reaction wells 160 such as from about 1 to about 300, about 1 to about 250, about 1 to about 200, about 1 to about 150, about 1 to about 100, about 1 to about 50, about 1 to about 40, about 1 to about 30, about 1 to about 20, about 1 to about 10, or about 1 to about 5.

[0049] Each reaction well 160 further includes a reagent for water quality analysis. The regent may be the same or different in each well 160. In some embodiments, the reagent comprises a nutrient and optionally an inhibitor. Each well 160 may have the same nutrient or a different nutrient. Further, each well 160 may have the same inhibitor or a different inhibitor (or no inhibitor at all).

[0050] Nutrients include methylumbelliferyl derivatives, such as MUG and 4- methylumbelliferyl-|3-D-glucoside, in addition to, for example, ONPG. Other nutrients may be selected by one of ordinary skill in the art based on the target bacteria. For example, one of ordinary skill in the art can determine nutrients capable of producing a fluorescent signal when metabolized by an enzyme of the target bacteria.

[0051] Nutrients may be selected such that they are specific to the target bacteria. In some embodiments, the nutrient fluoresces when metabolized by an enzyme of the target bacteria. In certain embodiments, the nutrient is hydrolyzed by the enzyme and the reaction product emits a fluorescent signal. The enzyme used in the presently disclosed methods may be any enzyme of the target bacteria. For example, E. coli bacteria use [l-glucuronidase to metabolize MUG and ONPG. Coliform use P-galactosidase to metabolize MUG and ONPG. P-glucuronidase and P- galactosidase are two illustrative examples of enzymes that can be used in connection with the presently disclosed devices and methods.

[0052] In some embodiments, tests conducted using the presently disclosed microfluidic device can be conducted in accordance with one or more of the steps set forth in “Evaluation of Enterolert for Enumeration of Enterococci in Recreational Waters,’’ G.E. Budnick et al., Applied and Environmental Microbiology, Oct. 1996, Vol. 62, No. 10, p. 3881-3884, the contents of which are incorporated into the present disclosure in their entirety.

[0053] Inhibitors may be present in the reagent if it would be desirable to inhibit growth of non-target bacteria so that only the target bacteria would be able to grow. Inhibitors include, but are not limited to, a salt, a sugar, a phenol, or any combination thereof.

[0054] Reagents, nutrients and inhibitors can be selected from those found in, for example, the Colilert™ and Enterolert™ products sold by IDEXX.

[0055] The sample board 104 may be embodied as a printed circuit board (PCB) and / or other integrated electronic device configured to perform the operations described herein. Illustratively, the sample board 104 includes a controller 140, one or more UV light-emitting diodes (LEDs) 142, an ammeter 144 or other current sensor, and multiple photodiodes 146. The UV LEDs 142 may be specific to the excitation frequency of the reaction product produced by the enzyme / nutrient interaction. The illustrative sample board 104 includes a single photodiode146 for each reaction well 160 of the sample tray 106 and sufficient UV LEDs 142 to illuminate the reaction wells 160. For example, in the illustrative embodiment in which the sample tray 106 includes 48 reaction wells 160, the sample board 104 may include 48 photodiodes 146 and 110 UV LEDs 142. Of course, it should be understood that in other embodiments the sample board 104 may include a different number and / or arrangement of fluorescence excitation sources 142 and / or fluorescence emission sensors 146. For example, in some embodiments, rather than photodiodes 146, the sample board 104 may include one or more phototransistors, photoresistors, or other light sensors.

[0056] Referring now to FIG. 2, one illustrative embodiment of the sample board 104 is shown. The sample board 104 includes a substrate 200, which is illustratively a printed circuit board (PCB). Attached to the substrate 200 is a control system 202, which illustratively includes the controller 140 and the ammeter 144. In the illustrative embodiment, the controller 140 is an Arduino microcontroller. In other embodiments, the controller 140 may be embodied as a microcomputer or single-board computer such as a Raspbery Pi, or any other microcontroller, controller, or control circuit. Further, although illustrated as including an ammeter 144 coupled to the sample board 104, in some embodiments the sample board 104 may be coupled to an off- board ammeter such as a 6485 Keithley pico-ammeter. Additionally, the control system 202 may further include one or more additional component such as integrated circuits, digital logic components, discrete components, or other components. One potential embodiment of the control system 202 is further described below in connection with FIG. 4.

[0057] As shown in FIG. 2, further attached to the substrate is a grid 204 of photodiodes 146. The illustrative photodiodes 146 are arranged in a rectangular grid 204 having a pitch distance 206 that separates each of the photodiodes 146. Multiple UV LEDs 142 are also attached to the substrate 200. The UV LEDs 142 surround the photodiodes 146. For example, each photodiode 146 may be surround on four sides by UV LEDs 142 as shown in FIG. 2.

[0058] Referring now to FIG. 3, the sample tray 106 may be placed on top of the sample board 104. As shown, the sample tray 106 includes an impermeable backing layer 300, which seals an aqueous medium 302 (e.g., fresh water or marine water) within each reaction well 160. The reaction wells 160 are arranged in a rectangular grid 304 on the sample tray 106, extending outward from the backing layer 300 toward the sample board 104. The reaction wells 160 in the rectangular grid 304 are separated by the pitch distance 206, which is the same pitch distance 206 separating the photodiodes 146. Accordingly, and as shown in FIG. 3, when the sample tray 106 is positioned over the sample board 104, each of the photodiodes 146 is positioned under a corresponding reaction well 160. Additionally, when the sample tray 106 is positioned over thesample board 104, the UV LEDs 142 are positioned between and surround the reaction wells 160. In some embodiments, the UV LEDs 142 may extend into the space separating the reaction wells 160 as shown in FIG. 3. In some embodiments, each photodiode 146 may be included within or otherwise positioned relative to one or more walls, a hood, a surround, or other light shade that blocks stray light, such as fluorescence from reaction wells 160 other than the corresponding reaction well 160 for that photodiode 146.

[0059] Referring now to FIG. 4, schematic diagram 400 illustrates one potential embodiment of the control system 202. As shown, the control system 202 includes the controller 140 of the sample board 104, which is coupled to the computer device 102. The control system 202 further includes multiple 2x1 multiplexers 404. Illustratively, the control system 202 includes one multiplexer 404 for each photodiode 146 and, thus, includes 48 multiplexers 404. Each multiplexer 404 includes an input coupled to an output of the corresponding photodiode 146 and two outputs that are coupled to ground and to the ammeter 144, respectively.

[0060] As shown in FIG. 4, for controlling and addressing the multiplexers 404, the multiplexers 404 are organized into six groups of eight multiplexers each, labeled multiplexers 404a through 404h. Each of the six groups of multiplexers is coupled to a corresponding 3x8 decoder 402a through 402f. The controller 140, in turn, is coupled to the six decoders 402a through 402f. In use, as described further below, the controller 140, using the decoders 402, may configure the multiplexers 404 to connect one of the photodiodes 146 to the ammeter 144 for current measurement. While a selected photodiode 146 is connected to the ammeter 144 via the corresponding multiplexer 404, the remaining other photodiodes 146 are connected to ground via their corresponding multiplexers 404. The arrangement illustrated in FIG. 4 allows for successful current isolation of the selected photodiode 146 while keeping all diodes conducting through the measurement and thus avoiding leakage current generation.

[0061] Referring now to FIGS. 5 and 6, in some embodiments the system 100 may be used to perform a method 500 for water quality analysis. The method 500 begins in block 502, in which the sample tray 106 is filled with water or other aqueous medium to be tested, such as fresh water (lake water, river water) or marine water (salt water, sea water, ocean water). For example, as described above, a 100 mL sample may be used to fill 48 reaction wells 160 of the sample tray 160. The aqueous medium may include, for example, a target bacteria, such as Enterococcus faecalis, total coliforms, fecal coliforms, fecal streptococci, Enterococci, and / or Escherichia coli. The reaction wells 160 are also filled (or may be pre-filled) with a reagent, which may include a nutrient and / or an inhibitor. As described above, within the reaction wells 160, a reaction may occur between the nutrient and an enzyme of the target bacteria. A productof that reaction provides a fluorescent signal. In block 504, the sample tray 106 is sealed. For example, a polymer, foil, or other impermeable layer may be attached to the sample tray 106, covering the reaction wells 106.

[0062] In block 506, the sample tray 106 is positioned on the sample board 104. When the sample tray 106 is positioned on the sample board 104, the UV LEDs 142 are capable of illuminating the interior of each reaction well 160, for example by shining through transparent walls of each reaction well. Similarly, the photodiodes 146 are capable of sensing fluorescence (e.g., light emitted at a particular wavelength) from within the reaction wells 160. In block 508, each photodiode 146 is positioned under a particular reaction well 160. In block 510, the UV LEDs 142 surround each reaction well 160. For example, the photodiodes 146 and the UV LEDs 142 may be positioned relative to the reaction wells 160 as shown in FIG. 3 and as described above. When the sample tray 106 is positioned on the sample tray 104, the assembly may be placed on the heater 108 or otherwise further prepared for analysis. Additionally or alternatively, although described as the sample tray 106 being positioned over the sample board 104, it should be understood that in other embodiments the sample board 104 and the sample tray 106 may be positioned together in any orientation such that the photodiodes 146 are capable of sensing fluorescence from within the reaction wells 160.

[0063] In block 512, the controller 110 maintains the sample tray 106 at a predetermined temperature using the heater 108. For example, the controller 110 may use a thermistor and a Peltier cell to maintain the sample tray 106 at a predetermined temperature, such as 41 °C, or otherwise about 40 °C. Of course, other heaters and / or predetermined temperatures may be used in other embodiments. For example, the device may be heated to a temperature of about 25 °C to about 55 °C, about 30 °C to about 50 °C, about 35 °C to about 45 °C, or about 39 °C, about 40 °C, about 41 °C, about 42 °C, or about 43 °C. In some embodiments, one or more functions of the controller 110 may be performed by a different device, such as the computing device 102 and / or the heater 108.

[0064] In block 514, the computing device 102 and / or the controller 140 of the sample board 104 periodically activates the UV LEDs 142 in order to illuminate the sample tray 106. For example, the UV LEDs 142 may be activated every 10 minutes or otherwise on a predetermined schedule. In some embodiments, the UV LEDs 142 may be activated continuously, responsively (e.g., in response to a request for measurement or other event), or otherwise activated.

[0065] In block 516, the computing device 102 and / or the controller 140 captures fluorescence data for each of the reaction wells 160 while one or more appropriate UV LEDs 142are activated. As described above, when the aqueous medium is illuminated with UV light by the UV LEDs 142, one or more reagents within the aqueous medium may fluoresce in the presence of a target bacteria or other pathogen. For example, when 4-methylumbelliferyl-P-D- glucoside is hydrolyzed by an enzyme of the bacteria, it produces 4-methylumbelliferone, which excites at about 365 nm and emits at about 465 nm. The fluorescence data may be embodied as digital or analog signals indicative of the intensity of light received by the photodiode 146 for each reaction well 160. The controller 140 may sequentially capture fluorescence data for each reaction well 160 by measuring data (e.g., measured electrical current) from the corresponding photodiode 146. In block 518, the controller 140 selects the photodiode 146 that corresponds with a particular reaction well 160. As shown in FIG. 4, the controller 140 may connect the selected photodiode 146 to the ammeter 144 using the associated multiplexer 404 and may connect all other non-selected photodiodes 146 to ground using their associated multiplexers 404. In block 520, the controller 140 measures the current generated by the selected photodiode 146 using the ammeter 144. Additionally or alternatively, in some embodiments, the ammeter 144 may store the current measurement for later processing by the controller 140 and / or the computing device 102. The controller 140 continues to sequentially select each photodiode 146 for current measurement until current measurements for all reaction wells 160 have been captured.

[0066] In block 522, the computing device 102 and / or the controller 104 determines whether sufficient time has elapsed in order to determine a water quality metric. As described above, in some embodiments, the sample tray 106 may be incubated for more than 3 hours, more than 6 hours, more than 12 hours, more than 24 hours, between 12 and 24 hours, or between 12 and 48 hours in order to determine a water quality metric. The time for water quality measurement may be predetermined and / or determined based on one or more measurements. If the time for measurement has not elapsed, the method 500 loops back to block 512, in which temperature of the sample tray 106 is maintained and fluoresce data continues to be captured. If the time for measurement has elapsed, the method 500 advances to block 524, shown in FIG. 6.

[0067] Referring now to FIG. 6, in block 524 the computing device 102 predicts a number of positive wells 160 based on the measured fluorescence data. A positive reaction well 160 is a reaction well in which fluorescence is detected, which indicates that the target bacteria or other pathogen interacts with the reagent within the reaction well 160. In many embodiments, some but not all of the reaction wells 160 will display fluorescence, and the number of such positive wells 160 may be used to determine a water quality metric.

[0068] In some embodiments, in block 526, the computing device 102 identifies an inflection point in the fluorescence data. Fluorescence data associated with bacterial growth may form a sigmoidal curve. Accordingly, the existence of an inflection point in the fluorescence data is indicative of bacterial growth within the corresponding reaction well 160. In some embodiments, in block 528 the computing device 102 compares a change in fluorescence signal to a predetermined threshold change in signal. In the presence of bacterial growth, the fluorescence data may display a small, but positive rate of change, even for relative short incubation times (e.g., less than 10 hours). Accordingly, the computing device 102 may compare a total change in current compared against an initial value against a predetermined threshold change in current. Reaction wells 160 with total change in current greater than or equal to the threshold may be considered positive, and reaction wells 160 with total change in current less than the threshold may be considered negative. The predetermined threshold may be 200 nA, 20 nA, 10 nA, or another predetermined current value. Smaller threshold values may allow for earlier detection times. For example, with a threshold equal to 200 nA, positive wells may be predicted after about 11 hours, with a threshold equal to 20 nA, positive wells may be predicted after about 9 hours, and with a threshold equal to 10 nA, positive wells may be predicted after about 3.3 hours. Predictions with smaller threshold values may be affected by noise, and accordingly higher resolution current measurements (e.g., about 10-bit resolution) may be used to improve recognition.

[0069] Although illustrated as predicting the number of positive wells 160 by identifying an inflection point and / or comparing signal change to a predetermined threshold, it should be understood that in other embodiments different techniques may be used to predict the number of positive wells. For example, in an embodiment a trained machine learning model such as a multilayer perceptron neural network (MLPNN) or other artificial neural network may be used to predict the number of positive wells 160.

[0070] In block 530, the computing device 102 generates a water quality metric based on the number of predicted positive wells. In block 532, the computing device 102 computes a most probable number (MPN) as a function of the number of wells. The number of flouresence- positive wells is related to the MPN of contamination. The unit of contamination is Colony Forming Units (CFU) and is an estimate of the number of viable bacteria in the water sample. The MPN analysis uses an equation that relates the number of positive wells, the total volume of negative (non-fluorescent) wells, and the total sample volume. For example,MPN = number of positive wellsx / [(number of ml in negative wells)*(number of ml in all wells)].

[0071] If there are no negative wells, the interpretation is that the contamination is higher than the maximum detection limit of the test sample tray 106. This is merely one example of calculating MPN and one of ordinary skill in the art will understand that additional methods can be used, such as a serial dilution test (https: / / www.fda.gov / food / laboratory-methods- food / bamappendix-2-most-probable-number-serial-dilutions). In block 534, the computing device 102 may compute a confidence interval.

[0072] In block 536, the computing device 102 reports the water quality metric. The computing device 102 may display, store, transmit, or otherwise provide the water quality metric to a user and / or to another system for further analysis or processing. By identifying an inflection point and / or comparing signal change to a predetermined threshold, the computing device 102 is capable of predicting positive wells with sufficient accuracy after a shorter incubation period compared to other techniques. For example, MPN analysis may be performed in 6 hours or 12, which is an improvement over other analysis techniques. After reporting the water quality metric, the method 500 may loop back to block 502, in which additional water samples may be tested.

[0073] The foregoing may be better understood by reference to the following examples, which are intended for illustrative purposes and are not intended to limit the scope of the disclosure or its application in any way.EXAMPLES

[0074] In one non-limiting test carried out by the inventors, a Quanti-Tray 2000 sample tray 106 including 48 reaction wells 160 was loaded with an aqueous medium sample. Enterolert reagent was added to the reaction wells 160 of the sample tray 106, and the sample tray 106 was sealed. The sample tray 106 was placed on a prototype sample board 104, and the assembly was placed in a temperature chamber and held at 40 °C for a period of 24 hours. The sample board 104 was used to capture fluorescence data with a sample period of 10 minutes, as described above.

[0075] Referring now to FIG. 7, chart 700 shows illustrative experimental results that may be achieved with the system 100. The chart 700 shows measured photodiode 106 current against time for the illustrative experiment. Curves 702 represent measured current for 47 photodiodes 106 corresponding to 47 of the reaction wells 160. Curve 704 represents measured current for one of the photodiodes 106, which malfunctioned during the experiment. Accordingly, curve 704 can be disregarded due to the malfunction. As shown, the remaining curves 702 exhibit sigmoidal growth curves characteristic of bacteria. Each curve 702 exhibitsan inflection point 706 at about 11 hours. Accordingly, in the illustrative experiment, the system 100 may be used to determine a water quality metric after an incubation period of 11 hours, which is faster than a typical incubation period of 24 hours.

[0076] When using a change in current threshold as an alternative metric for discrimination, for a threshold of 200 nA, all expected wells were found to be positive after 11 hours, which is similar to discriminating based on inflection point. For a threshold of 20 nA, all expected wells were found to be positive after 9 hours. For a threshold of 10 nA, all expected wells were found to be positive after about 3.3 hours, but the number of positive wells fluctuated due to sensor noise.

[0077] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While this invention may be embodied in many different forms, there are described in detail herein specific preferred embodiments of the invention. The present disclosure is an exemplification of the principles of the invention and is not intended to limit the invention to the particular embodiments illustrated. In addition, unless expressly stated to the contrary, use of the term “a” is intended to include “at least one” or “one or more.” For example, “a nutrient” is intended to include “at least one nutrient” or “one or more nutrients.”

[0078] Any ranges given either in absolute terms or in approximate terms are intended to encompass both, and any definitions used herein are intended to be clarifying and not limiting. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Moreover, all ranges disclosed herein are to be understood to encompass any and all subranges (including all fractional and whole values) subsumed therein.

[0079] Any composition disclosed herein may comprise, consist of, or consist essentially of any element, component and / or ingredient disclosed herein or any combination of two or more of the elements, components or ingredients disclosed herein.

[0080] Any method disclosed herein may comprise, consist of, or consist essentially of any method step disclosed herein or any combination of two or more of the method steps disclosed herein.

[0081] The transitional phrase “comprising,” which is synonymous with “including,” “containing,” or “characterized by,” is inclusive or open-ended and does not exclude additional, un-recited elements, components, ingredients and / or method steps.

[0082] The transitional phrase “consisting of’ excludes any element, component, ingredient, and / or method step not specified in the claim.

[0083] The transitional phrase “consisting essentially of’ limits the scope of a claim to the specified elements, components, ingredients and / or steps, as well as those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0084] As used herein, the term "about" refers to the cited value being within the errors arising from the standard deviation found in their respective testing measurements, and if those errors cannot be determined, then "about" may refer to, for example, within 5% of the cited value.

[0085] Furthermore, the invention encompasses any and all possible combinations of some or all of the various embodiments described herein. It should also be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the invention and without diminishing its intended advantages. It is therefore intended that such changes and modifications be covered by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A system for water quality analysis, the system comprising: a sample board comprising (i) a substrate, (ii) a plurality of fluorescence emission sensors coupled to the substrate, wherein the fluorescence emission sensors are arranged in a grid defined by a predetermined pitch, and (iii) a plurality of fluorescence excitation sources coupled to the substrate, wherein each of the fluorescence excitation sources is positioned adjacent to a fluorescence emission sensor; a sample tray comprising a plurality of reaction wells arranged in a grid defined by the predetermined pitch, wherein each reaction well comprises a reagent to carry out a reaction, and wherein each reaction well is transparent; and a computing device coupled to the sample board and configured to (i) cause the plurality of fluorescence excitation sources to illuminate the plurality of reaction wells when each reaction well of the sample tray includes an aqueous medium and the sample board is positioned on the sample tray, (ii) detect a fluorescence signal from each of the plurality of reaction wells with a corresponding fluorescence emission sensor, wherein each fluorescence signal is indicative of whether fluorescent activity is detected in the corresponding reaction well, and (iii) determine a water quality metric based on the fluorescence signal from each of the plurality of reaction wells.

2. The system of claim 1, wherein: each fluorescence excitation source comprises an ultraviolet (UV) light-emitting diode (LED); and each fluorescence emission sensor comprises a photodiode.

3. The system of claim 2, wherein: the sample board further comprises a control system comprising a controller and an ammeter coupled to the controller; and to detect the fluorescence signal from each of the plurality of reaction wells comprises to:sequentially select, by the controller of the sensor board, a photodiode associated with each reaction well; and measure, by the controller, current from the photodiode using the ammeter in response to selection of the photodiode.

4. The system of claim 3, wherein: the control system of the sample board further comprises a plurality of multiplexers, wherein each multiplexer comprises an input coupled to an output of a photodiode, a first output coupled to ground, and a second output coupled to the ammeter; and to sequentially select the photodiode associated with each reaction well comprises to (i) selectively cause a first multiplexer of the plurality of multiplexers to connect the input of the first multiplexer to the second output of the first multiplexer and (ii) cause remaining multiplexers of the plurality of multiplexers to connect the input of each respective multiplexer to the first output of each respective multiplexer.

5. The system of any of claims 1-4, wherein to determine the water quality metric comprises to: determine a number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity; and determine a most probable number (MPN) metric as a function of the number of the reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity.

6. The system of claim 5, wherein to determine the number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity comprises to determine an inflection point for the corresponding fluorescence signal.

7. The system of claim 5, wherein to determine the number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity comprises, for each fluorescence signal, to determine a change in fluorescence signal, and to determine whether the change in fluorescence signal has a predetermined relationship with a predetermined threshold.

8. The system of claim 7, wherein the fluorescence signal comprises a current received from a photodiode, and wherein to determine whether the change in fluorescence signal has the predetermined relationship with the predetermined threshold comprises to determine whether a change in current is greater than a predetermined change in current.

9. The system of claim 8, wherein the threshold change in current comprises 200 nA, 20 nA, or 10 nA.

10. The system of any of claims 1-4, further comprising: a heater coupled to a controller, the controller configured to heat the sample tray and the sample board at a predetermined temperature when the sample board is positioned on the sample tray; wherein the computing device is further configured to: continue to heat the device, illuminate the plurality of reaction wells, and detect the fluorescence signal from each of the plurality of reaction wells over a first time period between about 3.3 hours to less than 24 hours; and determine the water quality metric based on the fluorescence signal from each of the plurality of reaction wells after expiration of the first time period.

11. The system of claim 10, wherein the predetermined temperature is between about20 °C to about 60 °C.

12. The system of any of claims 1-4, wherein the reagent comprises a nutrient and optionally an inhibitor.

13. The system of any of claims 1-4, wherein the sample tray is formed from a transparent, impermeable polymer, wherein the impermeable polymer is thermoplastic, thermoset, machinable, manufacturable, translucent, biocompatible, chemically inert, resistant to ultraviolet light, or any combination thereof.

14. A device, comprising: a substrate; a plurality of fluorescence emission sensors coupled to the substrate, wherein the fluorescence emission sensors are arranged in a grid defined by a predetermined pitch; a plurality of fluorescence excitation sources coupled to the substrate, wherein each of the fluorescence excitation sources is positioned adjacent to a fluorescence emission sensor; and a control system coupled to the fluorescence emission sensors and the fluorescence excitation sources, wherein the control system is configured to (i) activate the fluorescence excitation sources and (ii) receive a fluorescence signal from each of the fluorescence emission sensors while the fluorescence excitation sources are activated.

15. The device of claim 14, wherein: each fluorescence excitation source comprises an ultraviolet (UV) light-emitting diode (LED); and each fluorescence emission sensor comprises a photodiode.

16. The device of claim 15, wherein the control system comprises a control system comprising a controller and an ammeter coupled to the controller, wherein the controller is configured to:sequentially select, by the controller of the sensor board, a photodiode from the plurality of photodiodes; and measure, by the controller, current from the photodiode using the ammeter in response to selection of the photodiode.

17. The device of claim 16, wherein: the control system further comprises a plurality of multiplexers, wherein each multiplexer comprises an input coupled to an output of a photodiode, a first output coupled to ground, and a second output coupled to the ammeter; and to sequentially select the photodiode comprises to (i) selectively cause a first multiplexer of the plurality of multiplexers to connect the input of the first multiplexer to the second output of the first multiplexer and (ii) cause remaining multiplexers of the plurality of multiplexers to connect the input of each respective multiplexer to the first output of each respective multiplexer.

18. A method for water quality analysis, the method comprising: loading an aqueous medium into a plurality of reaction wells of a sample tray, wherein each reaction well comprises a reagent to carry out a reaction, and wherein each reaction well is transparent; sealing the aqueous medium in the plurality of reaction wells; positioning a sample board on the sample tray after sealing the aqueous medium, wherein the sample board comprises a plurality of fluorescence excitation sources and a plurality of fluorescence emission sensors, and wherein when positioned on the sample tray, each fluorescence emission sensor is positioned to sense emissions from a corresponding reaction well; heating the sample tray and the sample board at a predetermined temperature; illuminating each of the plurality of reaction wells of the device with the plurality of fluorescence excitation sources;detecting a fluorescence signal from each of the plurality of reaction wells with a corresponding fluorescence emission sensor of a plurality of fluorescence emission sensors, wherein each fluorescence signal is indicative of whether fluorescent activity is detected in the corresponding reaction well; and determining, by a computing device, a water quality metric based on the fluorescence signal from each of the plurality of reaction wells.

19. The method of claim 18, wherein each fluorescence excitation source comprises an ultraviolet (UV) light-emitting diode (LED), and wherein each fluorescence emission sensor comprises a photodiode.

20. The method of claim 19, wherein detecting the fluorescence signal from each of the plurality of reaction wells comprises: sequentially selecting, by a controller of the sample board, a photodiode associated with each reaction well; and measuring, by the controller, current from the photodiode using an ammeter in response to selecting the photodiode.

21. The method of claim 20, wherein sequentially selecting the photodiode comprises (i) causing a multiplexer coupled to the photodiode to output current from the photodiode to the ammeter and (ii) causing a multiplexer coupled to each of the remaining photodiodes to output current to ground.

22. The method of any of claims 18-21, wherein determining the water quality metric comprises: determining a number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity; anddetermining a most probable number (MPN) metric as a function of the number of the reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity.

23. The method of claim 22, wherein determining the number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity comprises determining an inflection point for the corresponding fluorescence signal.

24. The method of claim 22, wherein determining the number of the plurality of reaction wells for which the corresponding fluorescence signal is indicative of detected fluorescent activity comprises, for each fluorescence signal, determining a change in fluorescence signal, and determining whether the change in fluorescence signal has a predetermined relationship with a predetermined threshold.

25. The method of claim 24, wherein the fluorescence signal comprises a current received from a photodiode, and wherein determining whether the change in fluorescence signal has the predetermined relationship with the predetermined threshold comprises determining whether a change in current is greater than a predetermined change in current.

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