Systems and methods for voltammetric detection

Abstract

A cartridge for voltammetric detection of a fluid parameter in a fluid sample is provided. The cartridge includes a sample reservoir including two compartments separated by a barrier fluid. Each compartment contains a chemical compound to facilitate voltammetric detection of the fluid parameter. A fluid collection device containing the fluid sample can be received by the sample reservoir, and the barrier can be penetrated by the fluid collection device, thereby causing the fluid sample to contact the two chemical compounds. When the fluid sample is introduced into the sample reservoir, the fluid parameter can be voltammetrically detected. Related components including the cartridge and methods for operating the cartridge are also described.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 791,352, filed on January 11, 2019; the entire contents of patent application No. 62 / 791,352 are hereby incorporated by reference into this application. Technical Field

[0003] The present disclosure relates generally to voltammetric detection systems, and in particular to voltammetric systems and methods for detecting fluid parameters in a fluid. Background Art

[0004] The following paragraphs provide background technology for the present disclosure. However, it is not considered that anything discussed herein is prior art or part of the knowledge of a person skilled in the art.

[0005] In many cases, it is necessary to detect chemicals in fluids. Thus, for example, water may contain chemicals that must be monitored and kept within certain tolerances in order to ensure a safe supply of domestic drinking water. Similarly, non-potable water, such as water used in industrial processes, must meet certain quality standards in order to be suitable for its intended use. Therefore, a variety of assays have been developed to monitor chemicals in water and other fluids.

[0006] One type of assay for detecting chemical species in fluids relies on the use of voltammetry. Typically, voltammetry involves the application of a voltage to a sample fluid containing electroactive chemical species, and the subsequent detection and evaluation of the current.

[0007] Ideally, the technology and system for detecting chemical substances in fluids are sensitive and fast. Although voltammetric techniques may be very sensitive, the execution of many voltammetric techniques requires the use of laboratory facilities, as well as the transportation and possible additional storage of fluid samples from the sampling location to the laboratory. The transportation of fluid samples may be time-consuming and expensive, and may include safety risks depending on the fluid. In addition, the fluid sample may experience changes in chemical composition during its transportation or storage, so when performing voltammetric analysis, the fluid sample may no longer represent the original composition of the chemical substance. In particular, voltammetric techniques involving biomolecules as sensors are rarely (if ever) known in the art, which allow voltammetric determination to be performed immediately at the position where the fluid sample is obtained.

[0008] Thus, although a variety of techniques are available for detecting chemicals in fluids, known techniques are not sufficiently effective. There is a continuing need in the art for improved processes for detecting chemicals, and in particular, for improved voltammetric systems. Summary of the invention

[0009] The following paragraphs are intended to introduce the reader to a more detailed description that continues but does not define or restrict the claimed subject matter of the present disclosure.

[0010] In one broad aspect, the present disclosure is directed to a system for voltammetric sensing of a fluid parameter in a fluid.

[0011] Therefore, on one hand, according to the teachings of this document, the present disclosure provides, in at least one embodiment, a sample analysis cartridge for voltammetric detection of a fluid parameter in a fluid sample, the cartridge comprising:

[0012] at least one sample analysis reservoir operable to receive an end of a fluid collection device for releasable collection of said fluid sample, said sample analysis reservoir comprising:

[0013] a first compartment, the first compartment containing an activating compound or an electroactive analyte, the electroactive analyte being activated by the activating compound to form an activated electroactive analyte when the activated compound is activated by the fluid parameter in the fluid sample, the first compartment having an opening at an upper end;

[0014] a second compartment, the second compartment comprising the electroactive analyte, the electroactive analyte being activated by the activated activating compound to form the activated electroactive analyte when the first compartment comprises the activating compound, or the second compartment comprising the activating compound when the first compartment comprises the electroactive analyte; and

[0015] a penetrable barrier disposed between the first compartment and the second compartment to fluidly separate the first compartment and the second compartment; and

[0016] A volt-ampere sensor is disposed at least partially within the second compartment.

[0017] In at least one embodiment, during use, (i) when the first compartment contains the activating compound and the sample fluid is in the sample analysis reservoir and in contact with the activating compound, the fluid parameters in the fluid sample can activate the activating compound, and when the barrier is subsequently ruptured, the activated activating compound can contact the electroactive analyte to form the activated electroactive analyte, which can then generate an electrical signal detected by the voltammetric sensor, thereby allowing the presence of the fluid parameter to be detected when a voltage is applied to the sample analysis reservoir, or (ii) when the first compartment contains the electroactive analyte, the sample fluid is in the sample analysis reservoir, and the barrier is subsequently ruptured, the fluid parameters in the fluid sample can activate the activating compound in the second compartment, and then the activated activating compound can contact the electroactive analyte to form the activated electroactive analyte, which can then generate an electrical signal detected by the voltammetric sensor, thereby allowing the presence of the fluid parameter to be detected when a voltage is applied to the voltammetric sensor.

[0018] In at least one embodiment, the cartridge may include a cartridge housing including a bottom housing portion and a top housing portion, the first compartment being disposed within the top housing portion and the second compartment being disposed within the bottom housing portion.

[0019] In at least one embodiment, the card box may include a card box housing, which includes a bottom housing portion, the penetrable barrier is arranged in the bottom housing portion to form the second compartment; and a top housing portion, which is slidably coupled to the bottom housing portion to form the first compartment.

[0020] In at least one embodiment, the card box may include a second penetrable barrier, which is disposed above the opening of the first compartment and includes a material that allows the end of the fluid collection device to penetrate it and be received by the first compartment to transfer the sample fluid therein when sufficiently forcefully engaged.

[0021] In at least one embodiment, the sample analysis reservoir may include a slot between the first compartment and the second compartment to slidably receive the penetrable barrier and separate the first compartment from the second compartment.

[0022] In at least one embodiment, the penetrable barrier separating the first compartment and the second compartment may include a depression, wherein the depression may contain the activating compound or the electroactive analyte.

[0023] In at least one embodiment, the penetrable barrier separating the first compartment and the second compartment can be made of a material that can be torn by the end of the fluid collection device.

[0024] In at least one embodiment, the penetrable barrier separating the first compartment and the second compartment can be made of a material that can be crushed by the end of the fluid collection device.

[0025] In at least one embodiment, the penetrable barrier separating the first compartment and the second compartment can be made of a material that can be pierced by the end of the fluid collection device.

[0026] In at least one embodiment, the cartridge can include two, three, four, five, or six sample reservoirs.

[0027] In at least one embodiment, at least two different sample reservoirs can be used to detect at least two different fluid parameters.

[0028] In at least one embodiment, at least two different sample reservoirs can be used to detect at least two identical fluid parameters.

[0029] In at least one embodiment, the activation compound can be a compound whose prolonged contact with the electroactive analyte can cause sufficient activation of the electroactive analyte to be voltammetrically detectable in the absence of fluidic parameters of the sample fluid.

[0030] In at least one embodiment, the prolonged contact can be from about 10 minutes to about 120 minutes.

[0031] In at least one embodiment, the activating compound can be an activating polypeptide.

[0032] In at least one embodiment, the activation compound can be an activation polypeptide formed by cells, wherein the cells contain a promoter that can be induced by the fluid parameters and controls the expression of the activation polypeptide.

[0033] In at least one embodiment, the cells may include microbial cells.

[0034] In at least one embodiment, the microbial cell can be a bacterial cell or a yeast cell.

[0035] In at least one embodiment, the microbial cells can be in liquid form.

[0036] In at least one embodiment, the microbial cells can be in a dried form.

[0037] In at least one embodiment, the microbial cells may be in the form of a gel.

[0038] In at least one embodiment, the microbial cells may include spore cells.

[0039] In at least one embodiment, the microbial cells may include Escherichia cells.

[0040] In at least one embodiment, the microbial cells may include Bacillus cells.

[0041] In at least one embodiment, the cell may include a microbial cell, and the activated polypeptide may be a hydrolase.

[0042] In at least one embodiment, the cell can include a microbial cell, and the activating polypeptide can be a phosphatase.

[0043] In at least one embodiment, the hydrolase can be selected from the group consisting of β-galactosidase, β-glucuronidase and β-glucosidase.

[0044] In at least one embodiment, the electroactive analyte may be chlorophenol red-β-D-pyranoside galactoside (CPRG), and the activating compound may be β-galactosidase, and when the electroactive analyte contacts the activating compound, chlorophenol red (CPR) is formed; or, the electroactive analyte may be p-nitrophenol-β-D-glucuronide (PNPG), and the activating compound may be β-glucuronidase, and when the electroactive analyte contacts the activating compound, p-nitrophenol (PNP) is formed; or, the electroactive analyte may be p-diphenol-β-D-pyranoside (PDPG G), and the activating compound may be β-glucosidase, and when the electroactive analyte contacts the activating compound, p-diphenol (PDP) is formed; the electroactive analyte may be p-aminophenol-β-pyranogalactoside (PAPG), and the activating compound may be β-galactosidase, and when the electroactive analyte contacts the activating compound, p-aminophenol (PAP) is formed; or, the electroactive analyte is p-aminophenyl phosphate (PAPP), and the activating compound is a phosphatase, and when the electroactive analyte contacts the activating compound, p-aminophenol (PAP) is formed.

[0045] In at least one embodiment, the fluid parameter may be a physical fluid parameter.

[0046] In at least one embodiment, the fluid parameter may be a chemical substance.

[0047] In at least one embodiment, the chemical substance may be an organic chemical compound.

[0048] In at least one embodiment, the chemical substance may be an inorganic chemical compound.

[0049] In at least one embodiment, the fluid parameter may be a toxic chemical.

[0050] On the other hand, the present disclosure provides, in at least one embodiment, a method for voltammetrically detecting a fluid parameter in a fluid, the method comprising:

[0051] Providing a fluid sample including a fluid parameter, the fluid sample being releasably collected in a fluid collection device; inserting an end of the fluid collection device into a cartridge, the cartridge comprising:

[0052] at least one sample analysis reservoir operable to receive the end of a fluid collection device, the sample analysis reservoir comprising:

[0053] a first compartment containing an activating compound, or an electroactive analyte, which can be activated by the activating compound to form an activated electroactive analyte when the activated compound is activated by the fluid parameter in the fluid sample, the first compartment having an opening at an upper end;

[0054] a second compartment, the second compartment comprising the electroactive analyte, the electroactive analyte being activated by the activating compound to form the activated electroactive analyte when the first compartment comprises the activating compound, or the second compartment comprising the activating compound when the first compartment comprises the electroactive analyte; and

[0055] a penetrable barrier disposed between the first compartment and the second compartment to fluidly separate the first compartment and the second compartment; and

[0056] a voltammetric sensor disposed at least partially within the second compartment;

[0057] moving the end of the fluid collection device into the first compartment and penetrating the barrier with the end of the fluid collection device to fluidically connect the first compartment and the second compartment and release the fluid sample from the fluid collection device into the sample analysis reservoir, causing the fluid parameters to activate the activating compound to produce the activated activating compound, which then contacts the electroactive analyte to form the activated electroactive analyte, which then contacts the voltammetric sensor;

[0058] applying a voltage to the voltammetric sensor;

[0059] detecting a current passing through the volt-ampere sensor; and

[0060] The detected current is compared to a threshold value to determine the presence of the fluid parameter in the fluid sample.

[0061] In at least one embodiment, when the first compartment contains the activating compound, the fluid sample can be released in the first compartment before penetrating the barrier.

[0062] In at least one embodiment, when the second compartment contains the activating compound, the fluid sample can be released in the second compartment after penetrating the barrier.

[0063] In at least one embodiment, the first compartment comprises a second penetrable barrier disposed over the opening, and the method comprises moving the fluid collection device with sufficient force to penetrate the second barrier and move into the first compartment.

[0064] In another aspect, the present disclosure provides, in at least one embodiment, a method for manufacturing a sample analysis cartridge for voltammetric detection of a fluid parameter in a fluid sample, the method comprising:

[0065] One or more sample reservoirs are formed for the sample analysis cartridge

[0066] Each sample storage device is manufactured as follows:

[0067] forming a bottom cartridge housing portion having a volt-ampere sensor therein;

[0068] placing an electroactive analyte or activating compound in the bottom cartridge housing portion;

[0069] forming a top cartridge housing portion;

[0070] forming a penetrable barrier to separate the bottom cartridge housing portion and the top cartridge housing portion;

[0071] placing an activating compound in the top cartridge housing portion when the electroactive analyte is placed in the bottom cartridge housing portion, or placing the electroactive analyte in the top cartridge housing portion over the penetrable barrier when the activating compound is placed in the bottom cartridge housing portion;

[0072] as well as

[0073] (a) placing the penetrable barrier on top of the bottom cartridge housing portion; and

[0074] slidably coupling the bottom cartridge housing portion and the top cartridge housing portion to form a cartridge; or

[0075] (b) slidably coupling the bottom cartridge housing portion and the top cartridge housing portion to form a cartridge, the cartridge comprising a slot between the bottom cartridge housing portion and the top cartridge housing portion to slidably receive the penetrable barrier; and slidably inserting the penetrable barrier into the slot.

[0076] In at least one embodiment, the method may further include providing a second penetrable barrier disposed over the opening of the top cartridge housing portion after slidably coupling the bottom cartridge housing portion and the top cartridge housing portion.

[0077] In at least one embodiment, the permeable barrier is formed to include one or more recesses to contain the activating compound or the electroactive analyte.

[0078] In at least one embodiment, the bottom housing compartment is formed to include one or more recesses to contain the electroactive analyte or the activating compound.

[0079] In at least one embodiment, the penetrable barrier is formed of a material that can be torn by the end of the fluid collection device.

[0080] In at least one embodiment, the method includes forming two, three, four, five or six sample reservoirs for the card box.

[0081] On the other hand, the present disclosure provides, in at least one embodiment, a voltammetric detection device for detecting a fluid parameter in a fluid sample contained in a sample analysis cartridge, the voltammetric detection device comprising:

[0082] at least one slot for releasably inserting the sample analysis cartridge of the present disclosure;

[0083] a voltage source configured to apply a voltage to a voltammetric sensor in a reservoir of the sample analysis cartridge;

[0084] a current detector for detecting a current passing through the sensor when the voltage is applied to the volt-ampere sensor; and

[0085] A controller is operably coupled to the voltage source and the current detector and is configured to control the operation of the volt-ampere detection device.

[0086] In at least one embodiment, the volt-ampere detection device may include two, three, four, five or six slots.

[0087] In at least one embodiment, the voltage detector may be operably coupled to a memory device.

[0088] On the other hand, the present disclosure provides a voltammetric detection component in at least one embodiment, the component comprising:

[0089] A volt-ampere detection device, the volt-ampere detection device comprising:

[0090] at least one slot for releasably inserting the sample analysis cartridge of the present disclosure;

[0091] a voltage source configured to apply a voltage to a voltammetric sensor in a reservoir of the sample analysis cartridge;

[0092] a current detector for detecting a current through the voltammetric sensor when the voltage is applied to the sensor; and

[0093] a controller operably coupled to the voltage source and the current detector and configured to control operation of the volt-ampere detection device; and

[0094] A fluid collection device is provided for releasably collecting a fluid sample for insertion into the sample analysis cartridge.

[0095] In another aspect, the present disclosure provides, in at least one embodiment, use of the sample analysis cartridge of the present disclosure for voltammetric detection of a fluid parameter in a fluid sample.

[0096] Other features and advantages of the present disclosure will become apparent through the following detailed description. However, it should be understood that the detailed description indicating the preferred embodiments of the present disclosure is given by way of illustration only, because through the detailed description, various changes and modifications within the spirit and scope of the present disclosure will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0097] The present disclosure is described in the paragraphs provided below in relation to the accompanying drawings by way of example. The drawings provided herein are for a better understanding of the example embodiments and to more clearly show how various embodiments may be implemented. In several views, which may be shown in different positions or from different angles, the same numerals represent the same or similar features. Therefore, by way of example only, Figure 2A , Figure 3B and Figure 3D Component 200 in FIG. 1 refers to the fluid collection device in each of these figures. The drawings are not intended to limit the present disclosure.

[0098] Figure 1Ais a perspective view of an example embodiment of a cartridge.

[0099] Figure 1B yes Figure 1A An exploded perspective view of the card box.

[0100] Figure 1C is along Figure 1A A vertical cross-sectional view of the card box taken along plane 1C.

[0101] Figure 1D is along Figure 1A Side view of the card box taken in plane 1D.

[0102] Figure 2A is a perspective view of an example embodiment of a fluid collection device.

[0103] Figure 2B is along Figure 2A A horizontal cross-sectional view of the fluid collection device taken along plane 2B.

[0104] Figure 2C is along Figure 2A A horizontal cross-sectional view of the fluid collection device taken along plane 2C.

[0105] Figure 3A is along Figure 1A A vertical cross-sectional view of the cartridge in a first state together with the fluid collection device, taken along plane 1C in FIG.

[0106] Figure 3B is along Figure 1A A vertical cross-sectional view of the cartridge in the second state together with the fluid collection device, taken on plane 1C in FIG.

[0107] Figure 3C yes Figure 3B A magnified perspective view of the area marked 3C in FIG.

[0108] Figure 3D is along Figure 1A A vertical cross-sectional view of the cartridge in the third state together with the fluid collection device, taken on plane 1C in FIG.

[0109] Figure 4A is a vertical cross-sectional view of another cartridge in a first state.

[0110] Figure 4B yes Figure 4B A vertical cross-sectional view of the card box in the second state.

[0111] Figure 4C is the cross-section of the barrier that can be penetrated.

[0112] Figure 5Ais a perspective view of a volt-ampere testing device with a cartridge inserted into a cartridge holder, showing the cartridge holder in an open position and the volt-ampere testing device in an open position.

[0113] Figure 5B yes Figure 5A A perspective view of a volt-ampere detection device with a cartridge inserted into a cartridge holder showing the cartridge holder in a closed position and the volt-ampere detection device in an open position.

[0114] Figure 5C yes Figure 5A A perspective view of a voltammetric test device showing the voltammetric test device in a closed position.

[0115] Figure 6 is a flow chart showing the use of Figure 1A -D card box and Figure 2A An exemplary embodiment of a method for voltammetric detection of a fluid parameter using a fluid collection device of -C.

[0116] Figure 7 is a block diagram illustrating an example embodiment of a configuration of a voltammetric detection apparatus.

[0117] Fig. 8A It is a top view of the volt-ampere detection device, wherein two cartridges are inserted into cartridge holders, showing two cartridge holders in an open position and two cartridge holders in a closed position, and the volt-ampere detection device in an open position.

[0118] Figure 8B 1 is a top view of the volt-ampere detection device, showing two cartridge supports in an open position and two cartridge supports in a closed position without a cartridge inserted, and the volt-ampere detection device in an open position.

[0119] Fig. 9 is a voltammogram illustrating experimental results obtained using an example embodiment of the cartridge and voltammometric detection apparatus of the present disclosure.

[0120] Fig.10 is another voltammogram illustrating experimental results obtained using an example embodiment of the cartridge and voltammometric detection apparatus of the present disclosure.

[0121] Fig.11 is a bar graph illustrating experimental results obtained using a cartridge containing a sample analysis reservoir including a single compartment and a voltammetric detection device.

[0122] Figures 12 to 17 is a voltammogram illustrating experimental results obtained using an example embodiment of the cartridge and voltammometric detection apparatus of the present disclosure.

[0123] Fig.18is another bar graph illustrating experimental results obtained using example embodiments of the cartridge and voltammetric detection apparatus of the present disclosure.

[0124] The drawings together with the following detailed description make clear to those skilled in the art how the present disclosure may be implemented in practice. DETAILED DESCRIPTION

[0125] Various processes, systems and compositions will be described below to provide at least one example of at least one embodiment of the claimed subject matter. The embodiments described below do not limit any claimed subject matter, and any claimed subject matter may cover processes, systems or compositions different from those described below. The claimed subject matter is not limited to any process, system or composition with all the features of the process, system or composition described below, or is not limited to the features common to multiple processes, systems, compositions or compositions described below. It is possible that the process, system or composition described below is not an embodiment of any claimed subject matter. Any subject matter disclosed in the process, system or composition described below that is not claimed in this document may be the subject of another protective instrument, for example, a consecutive patent application, and the applicant, inventor or right holder does not intend to abandon, waive or contribute to the public any such subject matter through the disclosure in this document.

[0126] As used herein and in the claims, singular forms such as "a", "an" and "the" include plural forms and vice versa unless the context clearly indicates otherwise. In this specification, unless otherwise indicated, the term "comprising" is used inclusively rather than exclusively, so that a specified integer or group of integers may include one or more other unspecified integers or groups of integers. The term "or" is inclusive unless modified, for example, to "either one or the other". The term "and / or" is intended to mean an inclusive or. For example, "X and / or Y" is intended to mean X or Y or both. As a further example, X, Y and / or Z are intended to mean X or Y or Z or any combination thereof.

[0127] When range is used in this article for physical properties such as molecular weight, or chemical properties such as chemical formula, all combinations and subcombinations of ranges and specific embodiments thereof are intended to be included. Except in the operating examples, or in the case of additional indications, all the numbers used herein representing the amount of ingredients or reaction conditions should be understood to be modified by the term "about" in all cases. When referring to a number or a numerical range, the term "about" means that the number or numerical range mentioned is an approximate value within the experimental variability (or statistical experimental error), so the number or numerical range can be changed between 1% and 15% of the specified number or numerical range, which is easily recognized in the context. In addition, the range of any value described herein is intended to specifically include the limit value of the range and any intermediate value or subrange within a given range, and all such intermediate values ​​and subranges are individually and specifically disclosed (for example, a range of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4 and 5). Similarly, other degree terms such as "substantially" and "approximately" as used herein mean a reasonable deviation amount of the modified term so that the final result will not change significantly. These terms of degree should be interpreted as including deviations of the modified term, for example up to 15%, if such deviation would not negate the meaning of the modified term.

[0128] For convenience, including for reference to the drawings, several directional terms are used herein, such as "above," "below," "lower," "upper," "inner," and "outer." Generally, the terms "upper," "above," "upwardly," and similar terms are used to refer to an upward direction or upper portion relative to a cartridge that remains generally upright, such as Figure 1A Similarly, the terms "lower", "below", "downward" and "bottom" are used to refer to a downward direction or lower portion relative to a card box that remains generally upright, such as Figure 1A The terms "inner" and "inwardly" are used herein to refer to directions closer to the radial center relative to the generally central longitudinal axis of the component, while the terms "outer" and "outwardly" refer to directions closer to the radial periphery relative to the generally central longitudinal axis of the component.

[0129] Unless otherwise defined, scientific and technical terms related to the formulations described herein should have the meanings commonly understood by those of ordinary skill in the art. The terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the claims.

[0130] All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically indicated to be incorporated by reference in its entirety.

[0131] Generally speaking, the sample analysis cartridges of the present disclosure may be used to detect fluid parameters, such as physical or chemical fluid parameters, present in a fluid sample.

[0132] In a broad sense, the sample analysis cartridge comprises a sample analysis reservoir in which a fluid sample can be received. The sample analysis reservoir contains an electroactive analyte that can be activated by a fluid parameter. It is noteworthy that the activation can be carried out via an intermediate activation compound that is also contained in the sample analysis reservoir. Also disposed in the cartridge is a voltammetric sensor. When the activated electroactive analyte contacts the voltammetric sensor, an electrical signal can be generated. The cartridge can be located in a voltammetric detection device so that an electrical signal indicating the presence of a fluid parameter in the fluid sample can be detected.

[0133] The cartridge of the present disclosure can be used in combination with a handheld voltammetric detection device. Therefore, the cartridge provided herein together with the voltammetric detection device and the fluid collection device can evaluate the presence of fluid parameters in a fluid sample at a location near the fluid sampling point. A challenge faced by many known voltammetric systems is that they can only be operated in a laboratory. Therefore, the fluid sample needs to be transported from the sampling point to the laboratory. The time delay between sampling and analysis may affect the accuracy of the analysis. In addition, depending on the sample fluid, there may be safety considerations associated with the transportation and storage of the sample fluid. In addition, the transportation and storage of the sample fluid may be costly.

[0134] In contrast, the cartridges of the present disclosure may be deployed at a fluid sampling point and may provide rapid analytical results at the sampling point without the need to transport or store the sample fluid, or without the need for additional laboratory equipment.

[0135] In addition, the cartridge of the present disclosure does not require reagent mixing involving multiple fluid transfer steps. Instead, the cartridge allows the user to obtain assay results using a single fluid transfer step, i.e., transferring the sample fluid to the cartridge.

[0136] Selected embodiments are described below with reference to the accompanying drawings.

[0137] In a general overview, Figures 1A-1D Several views of an example embodiment of the cartridge 100 are shown. Figures 2A-2C Several views of an example embodiment of a fluid collection device 200 for use in conjunction with the cartridge 100 are shown. Figures 3A-3D The cartridge 100 is shown operating in conjunction with a fluid collection device 200 . Figure 4A-4B Several views of another example cartridge 300 are shown. Figure 4C An example embodiment of a penetrable barrier is shown, which is a component of the cartridges described herein. Figures 5A-5C and Figures 8A-8B Several views of the cartridge 100 operating in conjunction with an example voltammetric detection apparatus 500 are shown. Figure 6A flow chart of an example method 600 of operating the cartridge 100 in conjunction with the fluid collection device 200 is shown. Figure 7 A block diagram showing an example configuration of a voltammetric detection device. Fig. 9 , 10 and 12-18 show graphs illustrating various experimental results obtained using cartridge 100, and Fig.11 A graph illustrating the results of using an alternative cartridge is shown, which will be further described in the Examples section of this disclosure.

[0138] First reference Figure 1A , which shows an example cartridge 100 for voltammetric detection of a sample fluid. The cartridge 100 includes a housing 102, which includes a top housing portion 105 and a bottom housing portion 110. The top housing portion 105 and the bottom housing portion 110 facilitate the manufacture and assembly of the cartridge 100, including the introduction of certain compounds into the cartridge 100, as will be further described below. The top housing portion 105 includes gripping members 120a and 120b to facilitate manipulation of the cartridge 100. The housing 102 is further configured to form three sample analysis reservoirs ( Figure 1A Each sample analysis reservoir has a top opening (not visible in Figure 1A 115a, 115b and 115c, so that each sample analysis storage is separated from the outside 130. It should be noted that in other embodiments, a card box including one or more sample analysis storages can be configured without a penetrable barrier covering the opening of such one or more sample analysis storages. In addition, in other embodiments, the card box of the present disclosure can include fewer (1 or 2) or more (for example, 4, 5, 6) sample analysis storages. Voltammetric sensors 125a, 125b and 125c are also shown, as shown in Figure 1B As can be better seen in FIG. 1 , each is associated with one of the three sample reservoirs. A variety of voltammetric sensors suitable for voltammetric detection can be used in various embodiments herein. Some example voltammetric sensors are described below.

[0139] Reference now Figure 1B , which shows an exploded view of an example cartridge 100. Figure 1A In addition to the various components shown in FIG. 1 , sample reservoirs 135a, 135b, and 135c disposed within the cartridge 100 and top openings 145a, 145b, and 145c of each of the sample reservoirs 135a, 135b, and 135c are also shown. It should be noted that when the cartridge 100 is assembled with the barriers 115a, 115b, and 115c in place (e.g., Figure 1A130 ), there is no fluid communication between the interior of the sample analysis reservoirs 135a, 135b, and 135c and the exterior 130. In addition, the interior housing walls 140a and 140b separate the sample reservoirs 135a and 135b and 135b and 135c from each other, respectively, so that there is no fluid communication between any of the sample reservoirs 135a, 135b, and 135c. Further shown is a penetrable barrier 190, which, when installed, divides the sample reservoirs 135a, 135b, and 135c into top and bottom portions, and between the sample reservoirs 135a, 135b, and 135c. Figure 1C 135a, 135b and 135c. The voltammetric sensors 125a, 125b and 125c are further shown, and it can now be better understood that the main parts of them are respectively disposed in the sample reservoirs 135a, 135b and 135c, and the bottom extends below the bottom housing portion 110. Located outside the voltammetric sensors 125a, 125b and 125c are conductive components 150a, 150b and 150c. When the cartridge 100 is assembled, the conductive components 150a, 150b and 150c are electrically coupled to the voltammetric sensors 125a, 125b and 125c, respectively. When used for voltammetric analysis, the conductive components 150a, 150b and 150c of the cartridge 100 can be electrically coupled to the electronic circuit of the voltammetric detection device. The voltammetric sensors 125a, 125b, and 125c and the conductive members 150a, 150b, and 150c are embedded together in the housing 102 to form at least a portion of the outer wall 185 of each of the sample reservoirs 135a, 135b, and 135c.

[0140] Reference now Figure 1C , which shows a cross-sectional view of an example cartridge 100. Figure 1A-1BIn addition to the several components shown in FIG. 1 , a sample reservoir 135 a is shown, which includes a top compartment 165 and a bottom compartment 170 defined by inner surfaces 155 and 160, respectively. As described above, the top compartment 165 and the bottom compartment 170 are fluidly separated from each other by a penetrable barrier 190. It should be noted that the geometry of the inner surfaces 155 and 160 can vary in different embodiments, for example, the top compartment 165 and the bottom compartment 170 can each be cylindrical, or approximately cylindrical, or conical, or approximately conical. In the illustrated embodiment, the top 170a of the bottom compartment 170 includes a generally inwardly and downwardly angled inclined surface 171, which can act as a stop to prevent the fluid collection device from reaching the bottom 170b of the bottom compartment 170, and further significantly prevent contact between the bottom surface 172 of the bottom 170b and the fluid collection device when the fluid collection device is received in the sample reservoir 135a, thereby facilitating the release of fluid from the fluid collection device, which is further described below. In general, the geometry of the inner surfaces 155 and 160 is designed to enable the fluid collection device to be received in the sample reservoir 135a to release the fluid sample, as described below. In addition, the curved inner surface area 160 extending downwardly and outwardly from the inwardly and downwardly angled surface 171 can facilitate receiving and accommodating the electroactive analyte in the bottom 170b of the bottom compartment 170, particularly when the electroactive analyte is provided in liquid form. In addition, it should be noted that the internal volumes of the top compartment 165 and the bottom compartment 170 can vary in different embodiments. In some embodiments, the relative volumes of the top and bottom compartments are somewhat similar, e.g., about 1:1, 1:2, 1:3, 1:4, or 4:1, 3:1, 2:1, or 1:1. In further embodiments, the total volume of sample reservoir 135a ranges from, e.g., about 250 μl to about 5,000 μl.

[0141] Reference now Figure 1D For further clarity, a cross-section of an example cartridge 100 is shown, indicating Figures 1A-1C Several components shown in .

[0142] Reference now Figure 2A -C, which shows an example embodiment of a fluid collection device 200 for use in conjunction with the cartridge of the present disclosure. The fluid collection device 200 includes a fluid reservoir portion 210 defined by an inner bore 202 and extending downwardly toward a tip portion 205, which includes a downwardly tapering tip reservoir portion 207 (see Figure 3A). The tip portion 205 can be received by a cartridge, as described below. The fluid reservoir portion 210 also includes a finger rest 212 and a plunger 214. The plunger 214 can move upward and downward within the hole 202 of the fluid reservoir portion 210. As will be readily understood by those skilled in the art, if the tip portion 205 is immersed in a fluid, the upward movement of the plunger 214 will allow the fluid to be drawn into the tip reservoir portion 207 of the tip portion 205 through the opening 215 and from there into the inner hole 202. Conversely, when the fluid is present in the inner hole 202, the downward movement of the plunger 214 will allow the fluid to move downward through the tip reservoir portion 207 and flow out through the opening 215 of the tip portion 205.

[0143] According to at least one example embodiment, a cartridge of the present disclosure, such as example cartridge 100, may be operated in conjunction with a fluid collection device 200, such as Figures 3A-3C shown.

[0144] Reference now Figure 3A , which shows an example cartridge 100 and an example fluid collection device 200 in a first state. There is no contact between the cartridge 100 and the fluid collection device 200. The fluid reservoir 210 of the fluid collection device 200 contains a fluid sample 219. The plunger 214, which extends downward to form a piston 216, extends upward in the first state shown, thereby allowing the fluid sample 219 to be contained in the fluid reservoir 210. The fluid collection device 200 can move in a downward direction d1 toward the penetrable barrier 115a of the sample reservoir 135a within the cartridge 100. In addition, the downward direction d2 indicates the downward movement of the plunger fluid 214 within the reservoir 210 of the fluid collection device 200 after the tip of the fluid collection device 200 has penetrated the barrier 115a.

[0145] Reference now Figure 3B-3C , which shows an example cartridge 100 and an example fluid collection device 200 in a second state. The fluid collection device 200 has now been sufficiently forcefully engaged with the penetrable barrier 115a to allow the tip 205 to penetrate the penetrable barrier 115a to extend into and be received by the top compartment 165 of the sample reservoir 135a. In addition, after the tip 205 of the fluid collection device 200 has been placed in the top compartment 165, the plunger 214 moves downward to cause the fluid sample 219 to be released into the top compartment 165 of the sample reservoir 135a. As shown in Figure 3CAs can be better seen in FIG. 1 , the fluid sample 219 is released from the tip reservoir portion 207 into the top compartment 165 (see: arrow F1). Contained in the top compartment 165 is an activation compound, which can be provided, for example, as dry particles or liquid deposited on the top surface of the barrier 190. Thus, when the fluid sample 219 in the top compartment 165 is released, the fluid sample 219 can contact the activation compound, and the fluid parameters can cause activation of the activation compound. Examples of activation compounds are described below.

[0146] refer to Figure 3D , which shows an example cartridge 100 and an example fluid collection device 200 in a third state. The fluid collection device 200 has now been sufficiently forcefully engaged with the penetrable barrier 190 to allow the tip 205 to penetrate the penetrable barrier 190 and move further into the cartridge 100 to be received by the bottom compartment 170 of the sample reservoir 135a. The plunger 214 has moved further downward, and an appropriate amount and possibly all of the fluid sample 219 has been released from the fluid collection device 200. Penetrating the penetrable barrier 190 has fluidically coupled the top compartment 165 and the bottom compartment 170. This has allowed the fluid sample 219 to enter the bottom compartment 170 of the sample reservoir 135a from the top compartment 165. The bottom compartment 170 contains an electroactive analyte, such as an electroactive analyte in liquid or particulate form, which has now been contacted with an activating compound to activate the electroactive analyte.

[0147] It is apparent that in the aforementioned exemplary operating embodiment, the fluid sample 219 can cause activation of the activation compound before fluidically coupling the top compartment 165 and the bottom compartment 170. In other operating embodiments, both the penetrable barriers 115a and 190 can be penetrated before the fluid sample 219 is released from the fluid collection device 200, in which case the fluid sample 219 is not released in the top compartment 165 of the fluid reservoir 205a, but in the bottom compartment 170 of the sample reservoir 135a. In this operating embodiment, the sample fluid, the activation compound, and the electroactive analyte can be in contact with each other more or less simultaneously. It should be noted that in such an operating embodiment, the electroactive analyte can be placed in the top compartment 165 and the activation compound can be placed in the bottom compartment 170.

[0148] It should be noted that the penetrable barrier 190 is preferably made of a tearable material, such as a foil or a film, to facilitate fluid coupling of the top compartment 165 and the bottom compartment 170 when it is penetrated. In contrast, the penetrable barrier 115a is preferably made of a substantially non-tearable and / or more durable material, but a pierceable material, such as rubber or silicone, so that after penetrating the barrier 115a, the possible influence of external parameters on the reaction between the fluid parameters and the activation compound, the reaction between the activation compound and the electroactive analyte, and the subsequent detection of the electroactivated analyte is limited because the pierceable material tightly surrounds the tip 205. Alternatively, in another embodiment, the penetrable barrier 190 can also be made of a breakable material, such as a breakable plastic or glass. In other alternative embodiments, the penetrable barrier 190 can be made of a substantially non-tearable and / or more durable material, but a pierceable material, such as rubber or silicone, such as tightly surrounding the tip 205.

[0149] It is also noted that in some embodiments, the penetrable barrier separating the top and bottom compartments of the fluid analysis reservoir can be manufactured to include a portion shaped to contain an activating compound, for example, in this regard, the penetrable barrier can include one or more depressions therein. An example of such a penetrable barrier is shown in Figure 4C . Therein is shown a penetrable barrier 415 which includes a recess 419 containing an activating compound 417. Similarly, in some embodiments, the bottom compartment 170, particularly the surface area 172 (see: Figure 1C ) can be manufactured to include a depression to contain the electroactive analyte. In an alternative embodiment, the depression 419 can contain the electroactive analyte, and the bottom compartment 170, particularly the surface area 172, can be manufactured to include a depression to contain the activating compound.

[0150] In short, a cartridge comprising one or more sample analysis storages has been provided. The sample analysis storage comprises a top compartment and a bottom compartment separated by a penetrable barrier 190, and can receive the end of a sample collection device comprising a sample fluid. The sample collection device can release the fluid in the top compartment 165, or release it into the bottom compartment 170 after penetrating the barrier 190. The top compartment 165 and the bottom compartment 170 respectively contain an activation compound and an electroactive analyte, or respectively contain an electroactive analyte and an activation compound. When the sample fluid is released in the sample storage 135a, 135b and / or 135c, the sample fluid, especially the sample fluid having a fluid parameter therein, can interact with the activation compound to activate it, and then the electroactive analyte can be activated. Then, the activated electroactive analyte can generate an electrical signal detected by the voltammetric sensor 125a, 125b and / or 125c, thereby allowing the presence of the fluid parameter to be detected when a voltage is applied to the voltammetric sensor across the sample analysis storage, as further described below.

[0151] Example embodiments of fluid parameters, activation compounds, and electroactive analytes will now be described.

[0152] Turning first to fluid parameters, it should be noted that the cartridges of the present disclosure can be used for voltammetric detection of any fluid parameter, including any parameter related to the physical properties of the fluid, i.e., physical fluid parameters, such as fluid turbidity, temperature, density, or viscosity, for example, and any parameter related to the chemical properties of the fluid, i.e., chemical fluid parameters, such as the presence of chemicals in the fluid, including, for example, organic molecules, biomolecules, or inorganic molecules. In an example embodiment, the fluid can be water, and the cartridge can be used to facilitate testing of water quality, such as by detecting the presence of at least one toxic chemical in a water sample; for example, mineral ions including, but not limited to, magnesium ions, potassium ions, and carbonate ions, and metal ions including, but not limited to, iron ions and lead ions; for example, and metalloid ions, but not limited to, arsenic ions.

[0153] In another example embodiment, the fluid can be water and the cartridge can be used to facilitate testing of turbidity. In yet another example embodiment, the fluid can be water and the cartridge can be used to facilitate testing of water quality, for example, by detecting the presence of contaminants (e.g., petroleum and petroleum derivatives) and toxins (e.g., biologically derived large polypeptide toxins (such as microcystins)) in water samples. The fluid parameters can vary, and those skilled in the art will readily appreciate that in different embodiments, the cartridges of the present disclosure can be used to detect a variety of fluid parameters in a variety of fluids, and activated compounds and electroactive analytes can be selected as needed to detect fluids and fluid parameters. It should also be noted that in embodiments in which it includes two or more sample analysis reservoirs, each sample analysis reservoir can be used to detect the same or different fluid parameters, or can be provided with the same or different sample fluids.

[0154] Turning now to the activation compound and electroactive analyte, including, as previously described, these components are contained in the top compartment or bottom compartment of the analysis reservoir of the cartridge, and in particular in a manner where the top compartment 165 and the bottom compartment 170 are fluidically separated before the cartridge is used for voltammetric analysis. Generally speaking, the activation compound and the electroactive analyte can be selected so that the fluid parameters of the sample fluid introduced into the fluid reservoir can activate the activation compound, and so that the activation compound can in turn activate the electroactive analyte, which can then be detected as an electric current by one of the voltammetric sensors 125a, 125b, and 125c corresponding to the sample / fluid analysis reservoir. In some embodiments, the top compartment 165 of the fluid reservoir can contain the activation compound and the bottom compartment 170 can contain the electroactive analyte. When the sample fluid in the top compartment 165 of the fluid reservoir is released, the activation compound will be activated. Subsequently penetrating the barrier 190 will cause the electroactive analyte to be activated by the activated activation compound. In other embodiments, the top compartment 165 of the fluid reservoir may contain an electroactive analyte, and the bottom compartment 170 may contain an activating compound. Only when the sample fluid collection device penetrates the barrier 190, the sample fluid is discharged, and the activating compound contacts the sample fluid and is activated, thereby being able to activate the electroactive analyte.

[0155] In certain embodiments, the activating compound can be a compound whose long-term contact with the electroactive analyte can cause the full activation of the electroactive analyte when there is no fluid parameter detecting the voltammetric signal. Although these compounds are suitable for voltammetric determination in principle, when they are together and contact each other for a long time, for example, more than one minute, one hour, one day, one week, one month or one year, or contact about 10 minutes to about 120 minutes before being used for voltammetric determination, they become unsuitable for voltammetric determination. It can be said that the long-term contact between these activating compounds and the electroactive analyte will destroy their use in voltammetric determination. Obviously, the current cartridge is configured so that the contact between the activating compound and the electroactive analyte can be avoided before performing voltammetric analysis, thereby preventing these components from being damaged. Therefore, the cartridge and the activated compound and the electroactive analyte included can be stored for a longer time, for example, at least about one month, at least about six months or at least about 12 months.

[0156] In one embodiment, the activating compound can be an activating polypeptide, such as but not limited to an enzyme.

[0157] In one embodiment, the activating compound can be an activating polypeptide formed by cells contained in the top compartment 165, the cells comprising a promoter that can be induced by fluid parameters and control the expression of the activating polypeptide. The cells can be living cells or dormant cells, such as microbial spore cells. The cells can also be microbial cells, such as yeast cells or bacterial cells, Escherichia cells or Bacillus cells, such as Escherichia coli cells, Bacillus subtilis cells and Bacillus thuringiensis cells.

[0158] In one embodiment, the cells may be dormant cells, and injection of fluid in the top compartment 165 causes the cells to exit dormancy and produce an activating compound.

[0159] In one embodiment, cells may be included in the top chamber 165 suspended in a liquid formulation (eg, water or a buffer).

[0160] In one embodiment, the cells can be included in the top compartment 165 in a dry formulation or a substantially dry formulation, which can be prepared by, for example, freeze-drying (ie, lyophilization) or air-drying a liquid cell suspension.

[0161] In one embodiment, cells can be included in the top compartment 165 in a gel formulation (eg, a gel matrix).

[0162] In one embodiment, the cell can be a microbial cell and the activating polypeptide can be a hydrolase.

[0163] In one embodiment, the cell can be a microbial cell and the activating polypeptide can be a phosphatase.

[0164] In one embodiment, the cell may be a microbial cell, and the activating polypeptide may be selected from the group consisting of β-galactosidase, β-glucuronidase, and β-glucosidase.

[0165] The promoter that can be induced by the fluid parameter and control the expression of the activated polypeptide can be an inducible promoter, such as a copper-sensitive promoter, including cusR promoter (PcusR), an iron-sensitive promoter, including fecA promoter (PfecA), a lead-sensitive promoter, including pbrA promoter (PpbrA), or an arsenic-sensitive promoter, including arsR promoter (ParsR). Other promoters that can be used are PhaA (pH-sensitive promoter), temperature-sensitive promoters, including heat shock promoters (such as Hsp70 or Hsp90 promoters), light-sensitive promoters (such as FixK2 promoters), or lac promoters (also called Plac) that can be induced by isopropyl-β-D-1-thiogalactopyranoside (IPTG).

[0166] In one embodiment, the electroactive analyte may be chlorophenol red-β-D-galactoside (CPRG) and the activating compound may be β-galactosidase, such that upon contact of the electroactive analyte with the activating compound, chlorophenol red (CPR) is formed.

[0167] In one embodiment, the electroactive analyte may be p-nitrophenol-β-D-glucuronide (PNPG) and the activating compound may be β-glucuronidase such that upon contact between the electroactive analyte and the activating compound, p-nitrophenol (PNP) is formed.

[0168] In one embodiment, the electroactive analyte may be p-diphenol-β-D-pyranoglucoside (PDPG) and the activating compound may be β-glucosidase such that upon contact between the electroactive analyte and the activating compound, p-diphenol (PDP) is formed.

[0169] In one embodiment, the electroactive analyte may be p-aminophenol-β-galactopyranoside (PAPG) and the activating compound may be β-galactosidase, such that upon contact between the electroactive analyte and the activating compound, p-aminophenol (PAP) is formed.

[0170] In one embodiment, the electroactive analyte may be p-aminophenyl phosphate (PAPP) and the activating compound may be a phosphatase such that upon contact between the electroactive analyte and the activating compound, p-aminophenol (PAP) is formed.

[0171] In an exemplary embodiment, the fluid parameter to be detected may be iron ions, and the activation compound may be formed by Escherichia cells (contained in the top compartment of the fluid analysis reservoir within the cartridge), the Escherichia cells comprising a fecA promoter that is induced by iron ions and controls the expression of β-galactosidase, which upon expression may activate p-aminophenol-β-galactoside (contained in the bottom compartment 170 of the fluid analysis reservoir within the cartridge) to form a voltammetrically detectable compound p-aminophenol (PAP).

[0172] In an exemplary embodiment, the fluid parameter to be detected may be xylene, and the activating compound may be formed by Bacillus thuringiensis cells (contained in the top compartment 165 of the fluid analysis reservoir within the cartridge), for example, in the form of dried dormant Bacillus thuringiensis spores that are capable of exiting dormancy upon contact with the fluid injected into the top compartment, the Bacillus thuringiensis cells comprising a Pu promoter that is inducible by xylene and controls the expression of β-glucosidase, which upon expression can activate p-diphenol-β-D-glucoside (contained in the bottom compartment of the fluid analysis reservoir within the cartridge) to form a voltammetrically detectable compound p-diphenol (PDP).

[0173] It should be noted that the above represents an example embodiment in which the activation compound, through prolonged contact with the electroactive analyte, can cause sufficient activation of the electroactive analyte, even in the absence of a fluid parameter in which a voltammetric signal is detected, that is, a small amount of β-galactosidase can be produced even in the absence of a fluid parameter, resulting in a low level of activation of the electroactive analyte. These low levels of β-galactosidase may be the result of a promoter, such as a "leakage" of the fecA promoter or the ArsR promoter. By separating the electroactive analyte from the activation compound, the cartridge of the present disclosure prevents the electroactive analyte from being activated by the result of promoter leakage (e.g., β-galactosidase) before performing a voltammetric measurement.

[0174] Turning now to the assembly of the cartridge of the present disclosure during manufacture, again referring to Figure 1A-C, in order to assemble the cartridge 100 including the activation compound and the electroactive analyte, in an exemplary embodiment, the following process may be followed. The top housing portion 105 and the bottom housing portion 110 of the cartridge 100 may be provided as separate components and placed within the fluid reservoirs 135a, 135b, and 135c together with appropriate amounts of the activation compound and the electroactive analyte. For example, the electroactive analyte may be easily introduced into the bottom compartment 170 of each fluid reservoir 135a, 135b, and 135c of the bottom housing portion 110 by depositing the particulate electroactive analyte into the bottom compartment 170. Thereafter, the barrier 190 may be added, and the activation compound may be introduced into the top compartment 165 of each fluid reservoir 135a, 135b, and 135c of the bottom housing portion 110, for example, by depositing the particulate electroactive analyte into the top compartment 165. It should be noted that in various embodiments, the activating compound and the electroactive analyte may be identical in each of the top compartments 165 and the bottom compartments 170 of the fluid reservoirs 135a, 135b, and 135c, for example, to allow the same activating compound and the electroactive analyte to be used to evaluate fluid samples obtained from multiple fluid sources. In other embodiments, different activating compounds and electroactive analytes may be included in each of the top compartments 165 and the bottom compartments 170 of the fluid reservoirs 135a, 135b, and 135c, for example, to allow voltammetric evaluation of fluid samples obtained from the same fluid source with respect to different fluid parameters. Finally, the top housing portion 105 may be slidably mounted on the bottom housing portion 110.

[0175] In at least one embodiment, the cartridge can be configured to include a releasable, penetrable barrier. Figure 4A and 4B As can be seen, the sample analysis reservoir 320 includes a slot 317 between the first compartment and the second compartment to slidably receive a penetrable barrier 315 to separate the top compartment 305 from the bottom compartment 310. The barrier 315 can be releasably inserted into and removed from the slot 317 of the cartridge 300.

[0176] This embodiment allows for the introduction of activating compounds or electroactive analytes after assembly of the top housing compartment 305 and the bottom housing compartment 310. In this embodiment, the penetrable barrier 315 may optionally include one or more recesses, e.g. Figure 4C shown.

[0177] According to an example embodiment, the cartridges of the present disclosure, such as the example cartridges 100 and 300, may be operated in conjunction with a voltammetric detection device 500, such as Figures 5A-5C shown.

[0178] The card box of the present disclosure can be inserted into a voltammetric detection device. Generally speaking, the voltammetric detection device is electronically configured to receive the card box of the present disclosure and apply a voltage to the voltammetric sensor of the card box. The voltammetric detection device is also generally electronically configured to detect the current passing through the sensor when the voltage is applied to the sensor.

[0179] Voltammetric techniques, methods and equipment are well known to those skilled in the art. In the following, some example techniques and equipment are described in general terms. It will be clear to those skilled in the art that the voltammetric detection equipment and techniques used in conjunction with the cartridges of the present disclosure may be varied and adjusted.

[0180] refer to Figure 6 , which shows a flow chart of an example embodiment of a method 600 for voltammetrically detecting a fluid parameter in a fluid sample according to the teachings herein. For ease of illustration, the method 600 involves Figure 1A -D card box and Figure 2A-C fluid collection device, but method 600 can be applied to alternative embodiments of the cartridge and fluid collection device. At action 602 of method 600, a fluid sample is collected from a source fluid in the fluid collection device 200. At action 604 of method 600, the end 205 of the fluid collection device 200 is sufficiently forcefully engaged with the penetrable barrier 115a to penetrate the penetrable barrier 115a and is received by the fluid reservoir 135a, particularly by the top compartment 165. At this point, in some embodiments, the cartridge 100 can be disengaged from the voltammetric detection device. In other embodiments, the cartridge 100 can be coupled to the voltammetric detection device, for example, by being inserted into a slot of the voltammetric detection device, the slot being configured to allow the cartridge 100 to be releasably inserted therein. At action 606 of method 600, the sample fluid is released from the fluid collection device 200 into the fluid reservoir 135a. In at least one embodiment, the sample fluid is released from the fluid collection device 200 in the top compartment 165, and then the end 205 engages with the penetrable barrier 190 with sufficient force to penetrate the penetrable barrier 190 to fluidly connect the top compartment 165 and the bottom compartment 170. In at least one embodiment, the end 205 can engage with the penetrable barrier 190 with sufficient force to penetrate the penetrable barrier 190 to fluidly connect the top compartment 165 and the bottom compartment 170, and then the sample fluid can be released from the fluid collection device 200. The release of the sample fluid from the fluid collection device 200 causes the fluid parameters in the sample to activate the activating compound contained in the top compartment 165, and then the activated activating compound activates the electroactive analyte contained in the bottom compartment 170. In some embodiments, mixing of the sample fluid, the activating compound, and the electroactive analyte can be further facilitated by shaking the cartridge 100. At act 608 of method 600, after the cartridge 100 has been coupled to the voltage source, a voltage is applied to the voltammetric sensor cartridge 100. At act 610 of method 600, a current is detected and cartridge 100 is also coupled to a current detector prior to applying a voltage. In at least one embodiment, the voltage source and current detector may be included in a voltammetric detection device. As previously described, in alternative embodiments, bottom compartment 170 may contain an activating compound and top compartment 165 may contain an electroactive analyte.

[0181] Turning now to voltammetric detection devices, a variety of voltammetric detection devices and voltammetric detection techniques may be used in accordance with the teachings herein. Figure 7, which shows an example block diagram of a configuration of a suitable voltammetric detection device 700, which includes a housing 714 and an output element 704, coupled to a voltammetric sensor 706. A voltage source 708 is coupled to the voltammetric sensor 706 and a controller 702 that can control the voltammetric sensor 706 to apply a voltage to the voltammetric sensor 706. When the voltammetric sensor 706 is in contact with an electroactive analyte, for example, when a fluid sample is introduced into a cartridge including the voltammetric sensor 706, as described above, a voltage is applied to the cartridge, which causes a current (i.e., an electrical signal) to pass through the voltammetric sensor 706 and be detected by a current detector 710, while the applied voltage can be detected by a voltage detector 712. The current detector 710 and the voltage detector 712 are also connected to the controller 702. The controller 702 can provide measured values ​​of the detected current and the applied voltage to the output element 704. In Figure 7 In the illustrated example embodiment 700, the voltage source 708, the current detector 710, the voltage detector 712, and the controller 702 are contained in a housing 714. The output element 704 coupled to the controller 702 is not contained in the housing 714 and may be coupled to the output element 704 via, for example, a cellular, Or WiFi communication protocol wirelessly coupled to the controller 702. In other embodiments, the output element 704 may also be included in the housing 714. The output element 704 may be a display, such as a liquid crystal display (LCD) or a light emitting diode (LED) display, or provided in the form of a series of LED indicators, for example, green, orange, red to indicate low, medium and high levels of liquid parameters. The controller 702 includes a processor and / or a detection circuit, and can process the detected current and the applied voltage to measure the fluid parameter, which can then be displayed by the output element 704. When detecting or measuring the fluid parameter, the detected current can be compared with a threshold value to determine the presence of the fluid parameter in the fluid sample. The controller 702 may be further coupled to a memory device or include a memory element (all not shown) to store one or more of the measured detected current, the measured detected applied voltage and the measured fluid parameter. The memory device may be removably coupled to the housing 714, for example, via a universal serial bus (USB) connection. It will be clear to those skilled in the art that various housing configurations can be assembled, including multiple housings that contain controllers, output elements, voltage sources, current detectors, voltage detectors and memory devices alone or together.

[0182] Therefore, as an example, a water sample suspected of containing a certain amount of arsenic can be collected by a fluid collection device and then introduced into a cartridge of the present disclosure by the fluid collection device, as described herein. The cartridge can include cells including a β-galactosidase controlled by an arsR promoter (ParsR) in the top compartment 165 of a given sample storage. The electroactive analyte aminophenol-β-pyranogalactoside (PAPG) can be included in the bottom compartment 170 of a given sample storage. When the water sample contacts the cells, in the presence of arsenic in the water sample, β-galactosidase can be produced, and when penetrating the barrier between the top compartment 165 and the bottom compartment 170, the electroactive analyte can be activated by β-galactosidase to form para-aminophenol (PAP), which can contact the voltammetric sensor 706. When the cartridge is placed in the voltammetric detection device, a voltage can be applied to the voltammetric sensor 706 by a voltage source 708 using a controller 702 and the applied voltage can cause a current to pass through the voltammetric sensor 706. Then, the current detector 710 can detect the flow of current, and the voltage detector 712 can detect the applied voltage. The controller 702 can provide the measured values ​​of the detected current and the applied voltage to the output element 704 for evaluation by the operator of the voltammetric detection device. A control cartridge, such as a cartridge containing a water sample known to be free of arsenic, can be used to compare and / or quantify the detected current relative to the current detected in the water sample suspected of containing arsenic to determine whether the detected current exceeds a threshold. In the case where the detected current exceeds the threshold represented by the control, it can be said that arsenic is present in the water sample. The controller 702 can be programmed to perform a comparison with the threshold and output the result to the output element 704.

[0183] The voltammetric detection device can be an operable device such that a voltage can be applied to the cartridge potentiostatically (i.e., at one voltage), incrementally at different selected voltages, such as in a square wave voltammetric manner, or in a cyclic voltammetric manner (i.e., linearly scanned over a defined voltage range). Also included are devices operable based on any voltammetric method, including but not limited to pulse voltammetry, linear sweep voltammetry, square wave voltammetry, chronoamperometry, step sweep voltammetry, and cyclic voltammetry, as well as variations or modifications thereof, such as differential pulse voltammetry, or wave-based voltammetry including a chronoamperometric step in the scan. Generally, according to the present invention, application of a voltage to a sample fluid can result in oxidation or reduction of an electroactive analyte and acquisition or release of electrons by the electroactive analyte or voltammetric sensor, which can be measured amperometrically in the form of an electric current.

[0184] The voltammetric sensor may generally include one or more working electrode assemblies, a reference electrode assembly, and a counter electrode assembly. The reference electrode may be any electrode that maintains a consistent voltage, and may be a suitable Ag / AgCl, a saturated calomel electrode (SCE), or a saturated sodium chloride calomel electrode (SSCE). The counter electrode may be, for example, a gold electrode, a platinum electrode, or a carbon electrode, such as a printed carbon, a glassy carbon, or a Vulcan carbon electrode. Considering the manufacturing cost, gold or glassy carbon electrodes may not be suitable for disposable cartridges. The composition of the working electrode may be different, and may be, for example, a gold, platinum, or carbon electrode, or a nanotube or nanoparticle electrode, or a graphene electrode. Examples of preferred electrode combinations include: 1) a gold working electrode, a reduced hydrogen reference electrode, and a platinum counter electrode; 2) a glassy carbon working electrode, a carbon counter electrode, and an Ag / AgCl reference electrode; 3) a platinum working electrode, a gold counter electrode, and an SCE reference electrode; and 4) a carbon working electrode, a carbon counter electrode, and an Ag / AgCl reference electrode. In general, given the general stability of the glassy carbon working electrode, a glassy carbon working electrode may be selected for preliminary exploratory studies. Considering that the manufacturing cost of these electrodes is more limited, carbon working electrode and counter electrode and Ag / AgCl reference electrode may be more preferably used for the manufacture of disposable cartridges. Electrodes can also be coated, for example, by a thiol self-assembled monolayer on a metal surface (e.g., a gold surface), and / or protective electrodes. For example, a carbon electrode can be protected by applying a phthalocyanine layer, by applying certain ions or metals (e.g., nickel), which can be dried on the electrode surface, or by platinum that can be plated on the surface. The aforementioned combination can also be applied. In at least one embodiment, screen-printed electrodes can be used, for example, using ceramic or plastic printed substrates, and paste-coated electrodes, for example, carbon paste for working electrodes and counter electrodes, and Ag / AgCl paste as a reference electrode.

[0185] Any stable reference electrode may be used to apply a voltage to the fluid, including, but not limited to, Ag / AgCl, a saturated calomel electrode (SCE), a saturated sodium chloride calomel electrode (SSCE), or a reduced hydrogen electrode (RHE), and the magnitude of the applied voltage may be selected between a first value that results in the production of oxygen from water at the positive end of the spectrum and a second value that results in the production of hydrogen from water at the negative end of the spectrum, and may depend on the specific electrode used. For example, a voltage in the range of 0-2.0V may be applied to the RHE reference electrode, or a voltage of -1 volt to +1 volt may be applied to the pseudo Ag / AgCl reference electrode. For example, when a constant potential voltage is applied, the detected amperage may range from 1 nA to 1 mA or more, or when cyclic voltammetry is used, the detected amperage may range from about 10 nA to 100 μA or more.

[0186] When voltage is applied to the sample fluid in the cartridge, the flow of electric current and its detection send a signal that there is a fluid parameter in the assay sample. On the contrary, when voltage is applied to the assay medium, there is no electrical signal or an electrical signal below a threshold value is detected indicating that there is no fluid parameter in the fluid sample. In this way, according to the present disclosure, the detection of electrical signals is related to the presence of fluid parameters in the fluid. The flow of electric current can be evaluated and / or quantified by the controller 702, and then output using a display device (e.g., a digital display device) as an output element 704, which is electronically configured to display the flow of the detected electric current measured. The display device can be included in the voltammetric detection device, or it can be coupled separately therewith. In at least one embodiment, the voltammetric detection device can also include an electronic memory component to store data associated with the detected current measured, the detected applied voltage measured, and the fluid parameter measured.

[0187] Reference now Figure 5A -C and Fig. 8A -B, which shows an example handheld voltammetric test device 500. The voltammetric test device 500 includes a reservoir 520 having an openable cover 525. A handle 540 allows the voltammetric test device 500 to be conveniently transported to a location, such as near a fluid sampling site. The cover 525 can be securely closed by clips 515a and 515b. The test device 500 also includes four cartridge holders 520a, 520b, 520c and 520d, each of which includes a slot that can releasably receive a different cartridge. In this regard, Figure 8B Slots 530c and 530d are shown without receiving a card cartridge, and Fig. 8A Slots 530c and 530d are shown in the figure, which receive card boxes 100a and 100b respectively. Each slot can also be covered by a hinged card box cover 510a, 510b, 510c and 510d. Fig. 8A and Figure 8B In FIG. 5 , hinged card box covers 510c and 510d are shown in a hinged open position. It should be noted that in addition to slots 530c and 530d, two slots are provided in the Fig. 8A and Figure 8B , as these slots are covered by hinged card box covers 510a and 510b. Figure 5A In the embodiment, only the slot 530d into which the cartridge 100b is inserted is visible, and the other three slots are covered by hinged cartridge covers 510a, 510b and 510c. The volt-ampere apparatus 500 also includes electronic circuitry that is required to perform volt-ampere detection using the cartridge 100, and the electronic circuitry may be, for example, Figure 7The voltammetric detection device 500 can be configured as shown. In different embodiments, the voltammetric detection device can include two, three, four, five, or six slots. For example, the slots can be configured to be the same or different, for example, each slot can be configured to accommodate the same cartridge, or a different cartridge, for example, a cartridge with a different voltammetric sensor. The voltammetric detection device 500 can be used to perform voltammetric detection on a fluid parameter present in a fluid sample introduced into a cartridge 100 inserted into one of the slots 530a, 530b, 530c, and 530d of the voltammetric detection device 500.

[0188] It should be noted that in some embodiments, slots 530a, 530b, 530c, and 530d and / or cartridges (e.g., 100a and 100b) can be color-coded. Similarly, penetrable barriers, e.g., 115a, 115b, and 115c of cartridge 100a, can be color-coded. Such color coding can facilitate analysis of different fluid parameters in different fluid reservoirs or cartridges. Thus, for example, each cartridge can include three different colored penetrable barriers, indicating intended use for detecting three different chemicals in each of the three reservoirs; alternatively, each of the four slots can be color-coded and paired with a similarly color-coded cartridge, indicating the intended use of the cartridge / slot for detecting four different chemicals.

[0189] As can now be appreciated, the cartridge of the present disclosure can be used for voltammetric detection of fluid parameters in a fluid sample. The cartridge can be used to detect fluid parameters in, for example, a water sample directly at the location where the sample is obtained, thereby evaluating water quality.

[0190] Of course, the above exemplary embodiments of the present disclosure are intended to be illustrative only and not restrictive. The described embodiments are susceptible to many modifications to composition, details, and order of operation. On the contrary, the present invention is intended to cover all such modifications within its scope, as defined by the claims, which should be given a broad interpretation consistent with the overall description.

[0191] Example

[0192] The following provides examples of further specific embodiments for carrying out the methods of the present disclosure, as well as embodiments of devices representing the present disclosure. It should be noted that this example is in further reference to using the devices and Figure 9-18 The results shown in are provided.

[0193] Example 1 - Cyclic voltammetry using β-galactosidase to generate PAP as an electroactive analyte

[0194] The cartridge 100 is configured to include a voltammetric sensor 706 including a working carbon electrode, a working carbon counter electrode, and an Ag / AgCl reference electrode. The cartridge 100 is then prepared to contain 0.16 ml of a 5 mM p-aminophenol-β-pyranogalactoside (PAPG) solution in a 0.2 M pH 7 sodium phosphate buffer in the bottom compartment 170 of the sample reservoirs 135a and 135b of the cartridge 100. The solution is allowed to stand to evaporate and form a crystalline residue. A total of 1 unit of β-galactosidase is introduced into the top compartment 165 of the sample reservoir 135a of the cartridge 100. No β-galactosidase is introduced into the top compartment 165 of the sample reservoir 135b of the cartridge 100. Using a fluid collection device substantially similar to fluid collection device 200, 800 μL of water is introduced into each of the two bottom compartments 170 of the sample reservoirs 135a and 135b of the cartridge 100 after piercing each penetrable barrier 190. The cartridge 100 is then briefly shaken to ensure mixing of the β-galactosidase, water, and PAPG, and the mixture is left to incubate, thereby allowing the PAPG to be degraded by the β-galactosidase to form para-aminophenol (PAP). Thereafter, the cartridge 100 is operably placed in the cartridge holder 520a of the voltammetric detection device 500, and a voltage from -0.4 v to +0.4 v is applied at 50 mv / s on the carbon counter electrode relative to the Ag / AgCl reference electrode in a cyclic voltammetric manner, and the measurements are made using the carbon working electrode relative to the reference electrode. As Fig. 9 As shown, voltammograms were prepared for the samples in each of the sample reservoirs 135a and 135b. It should be noted that in the sample reservoir 135b, which does not contain any β-galactosidase, no current changes were detected as a function of the changes in the cyclic applied voltage. In contrast, in the sample reservoir 135a, current changes were detected as a function of the changes in the applied voltage. The current changes can be explained as being caused by the presence of the electroactive analyte PAP in the sample reservoir 135a, which is formed by the enzymatic cleavage of PAPG by the enzyme β-galactosidase.

[0195] Example 2 - Cyclic voltammetry using protein phosphatase 1 (PP1) to produce PAP as an electroactive analyte

[0196] The cartridge 100 is configured to include a voltammetric sensor 706 including a working carbon electrode, a working carbon counter electrode, and an Ag / AgCl reference electrode. The cartridge 100 is then prepared to contain 0.16 ml of a 5 mM p-aminophenyl phosphate (PAPP) solution in a 0.2 M pH 7 sodium phosphate buffer in the bottom compartment 170 of the sample reservoirs 135a and 135b of the cartridge 100. The solution is allowed to stand to evaporate and form a crystalline residue. A total of 1 μg of protein phosphatase 1 (PP1) is introduced into the top compartment 165 of the sample reservoir 135a of the cartridge 100. No PP1 is introduced into the top compartment 165 of the sample reservoir 135b of the cartridge 100. Using a fluid collection device substantially similar to fluid collection device 200, 800 μL of water is introduced into each of the two bottom compartments 170 of the sample reservoirs 135a and 135b of the cartridge 100 after piercing each penetrable barrier 190. The cartridge 100 is then briefly shaken to ensure mixing of the PP1, water, and PAPP, and the mixture is left to incubate, thereby allowing the PAPP to be degraded by the PP1 to form PAP. Thereafter, the cartridge 100 is operably placed in the cartridge holder 520a of the voltammetric detection device 500, and a voltage from -0.4 v to +0.4 v is applied at 50 mv / s on the carbon counter electrode relative to the Ag / AgCl reference electrode in a cyclic voltammetric manner, and the measurements are made using the carbon working electrode relative to the reference electrode. Fig.13 As shown, voltammograms are prepared for the samples in each of the sample reservoirs 135a and 135b. It should be noted that in the sample reservoir 135b, which does not contain any PP1, no current change is detected as a function of the change in the cyclic applied voltage. In contrast, in the sample reservoir 135a, a current change is detected as a function of the change in the applied voltage. The change in current can be explained as being caused by the presence of the electroactive analyte PAP in the sample reservoir 135a, which is formed by the enzymatic degradation of PAPP by the enzyme PP1.

[0197] Example 3 - Evaluation of the voltammetric reaction time of bacterial cells expressing β-galactosidase to produce PAP as an electroactive analyte in a single compartment sample analysis reservoir

[0198] An alternative cartridge including a sample analysis reservoir is constructed, the sample analysis reservoir including a single compartment and no penetrable barrier. The alternative cartridge also includes a voltammetric sensor including a working carbon electrode, a working carbon counter electrode, and an Ag / AgCl reference electrode. The cartridge is then prepared to contain 0.16 ml of a 5 mM p-aminophenol-β-pyranogalactoside (PAPG) solution in a 0.2 M pH 7 sodium phosphate buffer in a single compartment of the sample analysis reservoir of the cartridge. The solution is allowed to stand for evaporation and to form a crystalline residue. Using a fluid collection device substantially similar to fluid collection device 200, a liquid culture of Escherichia coli containing an As(III)-inducible β-galactosidase expression plasmid with a total volume of 200 μL is introduced into the sample analysis reservoir. The cartridge is then briefly shaken to ensure that the E. coli culture and PAPG are mixed, and the mixture is allowed to stand for culture. Thereafter, the cartridge was operably placed in the cartridge holder of the voltammetric detection apparatus and a voltage from -0.4 V to +0.4 V was applied at 50 mv / s on the carbon counter electrode relative to the Ag / AgCl reference electrode in a cyclic voltammetric manner, and the measurements were made using the carbon working electrode relative to the reference electrode. The maximum value of the detected voltammetric current (related to the presence of p-aminophenol (PAP), an enzymatic degradation product of PAPG) was recorded at different incubation time points (t = 0; t = 10 minutes; t = 20 minutes; t = 30 minutes; t = 40 minutes; t = 50 minutes and t = 60 minutes). The bar graph of the results is shown in Fig.11 These results indicate that even in the absence of an inducing agent (i.e., As(III)), β-galactosidase exhibits catalytic activity after exposure to PAPG for a period of time, leading to the enzymatic degradation of PAPG and the formation of large amounts of voltammetrically detectable PAP.

[0199] Example 4 - Cyclic voltammetric detection of arsenite in water samples using bacterially expressed β-galactosidase controlled by an arsenite inducible promoter and using PAP as the electroactive analyte The cartridge 100 is configured to include a voltammetric sensor 706 including a working carbon electrode, a working carbon counter electrode, and an Ag / AgCl reference electrode. The cartridge 100 is then prepared to contain 0.16 ml of a 5 mM p-aminophenol-β-pyranogalactoside (PAPG) solution in 0.2 M pH 7 sodium phosphate buffer in each of the bottom compartments 170 of the sample reservoirs 135a, 135b, and 135c of the cartridge 100. The solution is left to evaporate and form a crystalline residue. A total volume of 200 μL of a liquid culture of E. coli containing an As(III)-inducible β-galactosidase expression plasmid was introduced into each of the top compartments 165 of the sample reservoirs 135a, 135b, and 135c of the cartridge 100. Using a fluid collection device substantially similar to the fluid collection device 200, three 800 μL water samples containing 0 ppb, 5 ppb, or 10 ppb As(III), respectively, were introduced into each of the bottom compartments 170 of the sample reservoirs 135a, 135b, and 135c of the cartridge 100 after piercing each penetrable barrier 190. The cartridge 100 was then shaken briefly to ensure mixing of the E. coli culture, water sample, and PAPG, and the mixture was left to incubate, thereby allowing As(III) to induce β-galactosidase expression and degradation of PAPG to form para-aminophenol (PAP). Thereafter, the cartridge 100 was operably placed in the cartridge holder 520a of the voltammetric testing apparatus 500 and a voltage of -0.4 V to +0.4 V was applied at 50 mV / s on the carbon counter electrode relative to the Ag / AgCl reference electrode in a cyclic voltammetric manner and the measurements were made using the carbon working electrode relative to the reference electrode. Voltammograms were prepared for each sample and displayed on Fig.12 It should be noted that the maximum value of the voltammetric current obtained using the water sample containing 10 ppb As(III) exceeds the maximum value of the voltammetric current obtained using the water sample containing 5 ppb As(III), which in turn exceeds the maximum value of the voltammetric current obtained using the water sample containing 0 ppb As(III).

[0200] Example 5 - Cyclic voltammetric detection of arsenite in water samples using bacterially expressed β-galactosidase controlled by an arsenite-inducible promoter and using CPR as an electroactive analyte

[0201] The cartridge 100 is configured to include a voltammetric sensor 706 including a working carbon electrode, a working carbon counter electrode, and an Ag / AgCl reference electrode. The cartridge 100 is then prepared to contain 0.16 ml of a 5 mM chlorophenol red-β-D-galactoside (CPRG) solution in a 0.2 M pH 7 sodium phosphate buffer in each bottom compartment 170 of the sample reservoirs 135 a, 135 b, and 135 c of the cartridge 100. The solution is allowed to stand to evaporate and form a crystalline residue. A liquid culture of Escherichia coli containing an As(III)-inducible β-galactosidase expression plasmid in a total volume of 200 μL is introduced into each top compartment 165 of the sample reservoirs 135 a, 135 b, and 135 c of the cartridge 100. Using a fluid collection device substantially similar to fluid collection device 200, three 800 μL water samples containing 0 ppb, 5 ppb or 10 ppb As(III), respectively, were introduced into each bottom compartment 170 of the sample reservoirs 135a, 135b and 135c of the cartridge 100 after piercing each penetrable barrier 190. The cartridge 100 was then briefly shaken to ensure mixing of the E. coli culture, water sample and CPRG, and the mixture was left to incubate, thereby allowing As(III) to induce β-galactosidase expression and CPRG degradation to form chlorophenol red (CPR). Thereafter, the cartridge 100 was operably placed in the cartridge holder 520a of the voltammetric detection device 500, and a voltage from 0 V to +1 V was applied at 50 mV / s to the carbon counter electrode relative to the Ag / AgCl reference electrode in a cyclic voltammetric manner, and the measurements were made using the carbon working electrode relative to the reference electrode. Voltammograms were prepared for each sample and displayed on Fig.13 It should be noted that the maximum value of the voltammetric current obtained using the water sample containing 10 ppb As(III) exceeds the maximum value of the voltammetric current obtained using the water sample containing 5 ppb As(III), which in turn exceeds the maximum value of the voltammetric current obtained using the water sample containing 0 ppb As(III).

[0202] Example 6 - Cyclic voltammetric detection of arsenite in water samples using bacterially expressed β-galactosidase controlled by an arsenite-inducible promoter and PNP as an electroactive analyte

[0203] The cartridge 100 is configured to include a voltammetric sensor 706 including a working carbon electrode, a working carbon counter electrode, and an Ag / AgCl reference electrode. The cartridge 100 is then prepared to contain 0.16 ml of a 5 mM p-nitrophenol-β-D-glucuronide (PNPG) solution in a 0.2 M pH 7 sodium phosphate buffer in the two bottom compartments 170 of the sample reservoirs 135 a and 135 b of the cartridge 100. The solution is allowed to stand to evaporate and form a crystalline residue. A liquid culture of Escherichia coli containing an As(III)-inducible β-galactosidase expression plasmid in a total volume of 200 μL is introduced into each of the top compartments 165 of the sample reservoirs 135 a and 135 b of the cartridge 100. Using a fluid collection device substantially similar to fluid collection device 200, two 800 μL water samples containing either 0 ppb or 5 ppb As(III), respectively, are introduced into each bottom compartment 170 of the sample reservoirs 135a and 135b of the cartridge 100 after piercing each penetrable barrier 190. The cartridge 100 is then briefly shaken to ensure mixing of the E. coli culture, water sample, and PNPG, and the mixture is left to incubate, thereby allowing the As(III) to induce β-galactosidase expression and degradation of PNPG to form para-nitrophenol (PNP). Thereafter, the cartridge 100 is operably placed in the cartridge holder 520a of the voltammetric detection device 500, and a voltage from -1 V to +0.4 V is applied at 50 mV / s to the carbon counter electrode relative to a Ag / AgCl reference electrode in a cyclic voltammetric manner, and the measurements are made using the carbon working electrode relative to the reference electrode. Voltammograms are prepared for each sample and displayed on Fig.14 Note that the maximum value of the voltammetric current obtained using the water sample containing 5 ppb As(III) exceeds the maximum value of the voltammetric current obtained using the water sample containing 0 ppb As(III).

[0204] Example 7 - Cyclic voltammetric detection of lead ions in water samples using bacterially expressed β-galactosidase controlled by a lead ion inducible promoter and PAP as an electroactive analyte

[0205] The cartridge 100 is configured to include a voltammetric sensor 706 including a working carbon electrode, a working carbon counter electrode, and an Ag / AgCl reference electrode. The cartridge 100 is then prepared to contain 0.16 ml of a 5 mM p-aminophenol-β-pyranogalactoside (PAPG) solution in a 0.2 M pH 7 sodium phosphate buffer in each bottom compartment 170 of the sample reservoirs 135a, 135b, and 135c of the cartridge 100. The solution is allowed to stand to evaporate and form a crystalline residue. A liquid culture of Escherichia coli containing a Pb(II)-inducible β-galactosidase expression plasmid in a total volume of 200 μL is introduced into each top compartment 165 of the sample reservoirs 135a, 135b, and 135c of the cartridge 100. Using a fluid collection device substantially similar to fluid collection device 200, three 800 μL water samples containing 0 ppb, 10 ppb or 20 ppb Pb(II), respectively, were introduced into each bottom compartment 170 of the sample reservoirs 135a, 135b and 135c of the cartridge 100 after piercing each penetrable barrier 190. The cartridge 100 was then briefly shaken to ensure mixing of the E. coli culture, water sample and PAPG, and the mixture was left to incubate, thereby allowing Pb(II) to induce β-galactosidase expression and degradation of PAPG to form para-aminophenol (PAP). Thereafter, the cartridge 100 was operably placed in the cartridge holder 520a of the voltammetric detection device 500, and a voltage from -0.4 V to +0.4 V was applied at 50 mV / s on the carbon counter electrode relative to the Ag / AgCl reference electrode in a cyclic voltammetric manner, and the measurements were made using the carbon working electrode relative to the reference electrode. Voltammograms were prepared for each sample and displayed on Fig.15 It should be noted that the maximum value of the voltammetric current obtained using the water sample containing 20 ppb Pb(II) exceeds the maximum value of the voltammetric current obtained using the water sample containing 10 ppb Pb(II), which in turn exceeds the maximum value of the voltammetric current obtained using the water sample containing 0 ppb Pb(II).

[0206] Example 8 - Square wave voltammetric detection of arsenite in water samples using bacterially expressed β-galactosidase controlled by an arsenite-inducible promoter and using PAP as an electroactive analyte

[0207] The cartridge 100 is configured to include a voltammetric sensor 706 including a working carbon electrode, a working carbon counter electrode, and an Ag / AgCl reference electrode. The cartridge 100 is then prepared to contain 0.16 ml of a 5 mM p-aminophenol-β-pyranogalactoside (PAPG) solution in a 0.2 M pH 7 sodium phosphate buffer in each bottom compartment 170 of the sample reservoirs 135a, 135b, and 135c of the cartridge 100. The solution is allowed to evaporate and a crystalline residue is formed. A liquid culture of Escherichia coli containing an As(III)-inducible β-galactosidase expression plasmid in a total volume of 200 μL is introduced into each top compartment 165 of the sample reservoirs 135a, 135b, and 135c of the cartridge 100. Using a fluid collection device substantially similar to fluid collection device 200, three 800 μL water samples containing 0 ppb, 5 ppb or 10 ppb As(III), respectively, were introduced into each bottom compartment 170 of the sample reservoirs 135a, 135b and 135c of the cartridge 100 after piercing each permeable barrier 190. The cartridge 100 was then briefly shaken to ensure mixing of the E. coli culture, water sample and PAPG, and the mixture was left to incubate, thereby allowing As(III) to induce β-galactosidase expression and degradation of PAPG to form para-aminophenol (PAP). Thereafter, the cartridge 100 was operably placed in the cartridge holder 520a of the voltammetric detection device 500, and a voltage of -0.4 V to +0.4 V was applied to the carbon counter electrode relative to the Ag / AgCl reference electrode in a square wave voltammetric manner, wherein the voltage step height was 5 mV, the pulse height was 25 mV, and the pulse width was 50 ms. The measurements were made using a carbon working electrode relative to a reference electrode. Voltammograms were prepared for each sample and displayed on Fig.16 It should be noted that the maximum value of the voltammetric current obtained using the water sample containing 10 ppb As(III) exceeds the maximum value of the voltammetric current obtained using the water sample containing 5 ppb As(III), which in turn exceeds the maximum value of the voltammetric current obtained using the water sample containing 0 ppb As(III).

[0208] Example 9 - Square Wave Voltammetric Detection of Lead Ions in Water Samples Using Bacterially Expressed β-Galactosidase Controlled by a Lead Ion Inducible Promoter and PAP as an Electroactive Analyte

[0209] The cartridge 100 is configured to include a voltammetric sensor 706 including a working carbon electrode, a working carbon counter electrode, and an Ag / AgCl reference electrode. The cartridge 100 is then prepared to contain 0.16 ml of a 5 mM p-aminophenol-β-pyranogalactoside (PAPG) solution in a 0.2 M pH 7 sodium phosphate buffer in each bottom compartment 170 of the sample reservoirs 135a, 135b, and 135c of the cartridge 100. The solution is allowed to evaporate and a crystalline residue is formed. A liquid culture of E. coli containing a Pb(II)-inducible β-galactosidase expression plasmid in a total volume of 200 μL is introduced into each top compartment 165 of the sample reservoirs 135a, 135b, and 135c of the cartridge 100. Using a fluid collection device substantially similar to fluid collection device 200, three 800 μL water samples containing 0 ppb, 10 ppb or 20 ppb Pb(II), respectively, were introduced into each bottom compartment 170 of the sample reservoirs 135a, 135b and 135c of the cartridge 100 after piercing each penetrable barrier 190. The cartridge 100 was then briefly shaken to ensure mixing of the E. coli culture, water sample and PAPG, and the mixture was left to incubate, thereby allowing Pb(II) to induce β-galactosidase expression and degradation of PAPG to form para-aminophenol (PAP). Thereafter, the cartridge 100 was operably placed in the cartridge holder 520a of the voltammetric detection device 500, and a voltage of -0.4 V to +0.4 V was applied to the carbon counter electrode relative to the Ag / AgCl reference electrode in a square wave voltammetric manner, wherein the voltage step height was 5 mV, the pulse height was 25 mV, and the pulse width was 50 ms. The measurements were made relative to a reference electrode using a carbon working electrode. Voltammograms were prepared for each sample and displayed on Fig.17 It should be noted that the maximum value of the voltammetric current obtained using the water sample containing 20 ppb Pb(II) exceeds the maximum value of the voltammetric current obtained using the water sample containing 10 ppb Pb(II), which in turn exceeds the maximum value of the voltammetric current obtained using the water sample containing 0 ppb Pb(II).

[0210] Example 10 - Using PAP as an electroactive analyte to evaluate the effect of cartridge storage time on bacterial cell expression of β-galactosidase

[0211] The three cartridges 100 were constructed to include a voltammetric sensor 706 including a working carbon electrode, a working carbon counter electrode and an Ag / AgCl reference electrode and stored for a storage period of 1 day, 3 days and 9 days, respectively. Each of the three cartridges 100 was then prepared to contain 0.16 ml of a 5 mM solution of p-aminophenol-β-pyranogalactoside (PAPG) in a 0.2 M pH 7 sodium phosphate buffer in each bottom compartment 170 of the sample reservoirs 135a, 135b and 135c of the cartridge 100. The solution was allowed to evaporate and a crystalline residue was formed. A liquid culture of Escherichia coli containing an As(III)-inducible β-galactosidase expression plasmid in a total volume of 200 μL was introduced into each top compartment 165 of the sample reservoirs 135a, 135b and 135c of each of the three cartridges 100. After the storage period, three 800 μL water samples containing 0 ppb, 5 ppb or 10 ppb As(III), respectively, were introduced into each bottom compartment 170 of the sample reservoirs 135a, 135b and 135c of the cartridge 100 using a fluid collection device substantially similar to the fluid collection device 200, after piercing each permeable barrier 190. Each of the three cartridges 100 was then briefly shaken to ensure mixing of the E. coli culture, water sample and PAPG, and the mixture was left to incubate, thereby allowing As(III) to induce β-galactosidase expression and degradation of PAPG to form para-aminophenol (PAP). Thereafter, each of the three cartridges 100 was operably placed in the cartridge holder 520a of the voltammetric detection device 500, and a voltage from -0.4 V to +0.4 V was applied at 50 mV / s on the carbon counter electrode relative to the Ag / AgCl reference electrode in a cyclic voltammetric manner, and the measurements were made using the carbon working electrode relative to the reference electrode. A voltammogram was prepared for each sample and the maximum value of the detected voltammogram current was recorded for each As(III) concentration at each storage period. A bar graph with the results is shown in Fig.18 It should be noted that for each storage period, i.e., 1 day, 3 days, and 9 days, the maximum voltammetric current detected using the water sample containing 10 ppb As(III) exceeded the maximum voltammetric current detected using the water sample containing 5 ppb As(III), and conversely, for each storage period, it exceeded the voltammetric peak value obtained using the water sample containing 0 ppb As(III). It should also be noted that the current detected after 9 days of storage was not significantly greater than the current detected after 1 day of storage, which reflects the lack of reaction between the microbial cells in each top compartment and the PAPG in the bottom compartment.

Claims

1. A sample analysis cartridge for voltammetric detection of fluid parameters in a fluid sample, characterized in that: The card box comprises: at least one sample analysis reservoir operable to receive an end of the fluid collection device for releasable collection of the fluid sample from the fluid collection device, the at least one sample analysis reservoir comprising: a first compartment, the first compartment containing an activation compound or an electroactive analyte, the electroactive analyte being activated by the activation compound to form an activated electroactive analyte when the activation compound is activated by the fluid parameter in the fluid sample, the first compartment having an opening at an upper end; a second compartment, the second compartment comprising the electroactive analyte, the electroactive analyte being activated by the activated activating compound to form the activated electroactive analyte when the first compartment comprises the activating compound, or the second compartment comprising the activating compound when the first compartment comprises the electroactive analyte; and a penetrable barrier disposed between the first compartment and the second compartment to fluidly separate the first compartment and the second compartment and further configured to be engageable by the fluid collection device; and A volt-ampere sensor is disposed at least partially within the second compartment.

2. The sample analysis cartridge according to claim 1, characterized in that: The sample analysis cartridge is configured such that (i) when the fluid sample is in the at least one sample analysis reservoir and in contact with the activating compound, the fluid parameters in the fluid sample activate the activating compound, and when the barrier is subsequently ruptured, the activated activating compound contacts the electroactive analyte to form the activated electroactive analyte, which in turn generates an electrical signal detected by the voltammetric sensor, thereby allowing the presence of the fluid parameter to be detected when a voltage is applied to the at least one sample analysis reservoir, or (ii) when the first compartment contains the electroactive analyte, the fluid sample is in the at least one sample analysis reservoir, and the barrier is subsequently ruptured, the fluid parameters in the fluid sample activate the activating compound in the second compartment, which then contacts the electroactive analyte to form the activated electroactive analyte, which in turn generates an electrical signal detected by the voltammetric sensor, thereby allowing the presence of the fluid parameter to be detected when a voltage is applied to the voltammetric sensor.

3. The sample analysis cartridge according to claim 1 or 2, characterized in that: The cartridge includes a cartridge housing including a bottom housing portion and a top housing portion, the first compartment being disposed within the top housing portion and the second compartment being disposed within the bottom housing portion.

4. The sample analysis cartridge according to claim 1 or 2, characterized in that: The cartridge includes a cartridge housing including a bottom housing portion within which the penetrable barrier is disposed to form the second compartment, and a top housing portion slidably coupled to the bottom housing portion to form the first compartment.

5. The sample analysis cartridge according to claim 1 or 2, characterized in that: The cartridge includes a second penetrable barrier disposed over the opening of the first compartment and comprising a material that allows an end of a fluid collection device to penetrate the barrier and be received by the first compartment upon sufficiently forceful engagement to deliver the fluid sample therein.

6. The sample analysis cartridge according to claim 1 or 2, characterized in that: The at least one sample analysis reservoir includes a slot between the first compartment and the second compartment to slidably receive the penetrable barrier and separate the first compartment from the second compartment.

7. The sample analysis cartridge according to claim 1 or 2, characterized in that: The penetrable barrier separating the first compartment and the second compartment comprises a recess, wherein the recess contains the activating compound or the electroactive analyte.

8. The sample analysis cartridge according to claim 1 or 2, characterized in that: The penetrable barrier separating the first compartment and the second compartment is made of a material that can be torn by the end portion of the fluid collection device.

9. The sample analysis cartridge according to claim 1 or 2, characterized in that: The penetrable barrier separating the first compartment and the second compartment is made of a material that can be crushed by the end of the fluid collection device.

10. The sample analysis cartridge according to claim 1 or 2, characterized in that: The penetrable barrier separating the first compartment and the second compartment is made of a material that can be pierced by the end of the fluid collection device.

11. The sample analysis cartridge according to claim 1 or 2, characterized in that: The cartridge comprises two, three, four, five or six sample reservoirs.

12. The sample analysis cartridge according to claim 11, characterized in that: The cartridge comprises at least two different sample reservoirs configured to detect at least two different fluid parameters.

13. The sample analysis cartridge according to claim 11, characterized in that: The cartridge contains at least two different sample reservoirs configured to detect the same fluid parameter.

14. The sample analysis cartridge according to claim 1 or 2, characterized in that: The sample analysis cartridge is configured such that the activating compound, when in prolonged contact with the electroactive analyte, causes sufficient activation of the electroactive analyte to be voltammetrically detectable in the absence of fluidic parameters of the fluid sample.

15. The sample analysis cartridge according to claim 14, characterized in that: The prolonged contact is 10 minutes to 120 minutes.

16. The sample analysis cartridge according to claim 1 or 2, characterized in that: The activating compound is an activating polypeptide.

17. The sample analysis cartridge according to claim 1 or 2, characterized in that: The activation compound is an activation polypeptide formed by cells contained in the first compartment, the cells comprising a promoter inducible by the fluid parameters and controlling the expression of the activation polypeptide.

18. The sample analysis cartridge according to claim 17, characterized in that: The cells include microbial cells.

19. The sample analysis cartridge according to claim 18, characterized in that: The microbial cells include bacterial cells or yeast cells.

20. The sample analysis cartridge according to claim 18, characterized in that: The microbial cells are in liquid form.

21. The sample analysis cartridge according to claim 18, characterized in that: The microbial cells are in a dried form.

22. The sample analysis cartridge according to claim 18, characterized in that: The microbial cells are in the form of a gel.

23. The sample analysis cartridge according to claim 18, characterized in that: The microbial cells include spore cells.

24. The sample analysis cartridge according to claim 18, characterized in that: The microbial cells include Escherichia cells.

25. The sample analysis cartridge according to claim 18, characterized in that: The microbial cells include Bacillus cells.

26. The sample analysis cartridge according to claim 17, characterized in that: The cell comprises a microbial cell, and the activated polypeptide is a hydrolase.

27. The sample analysis cartridge according to claim 17, characterized in that: The cell comprises a microbial cell, and the activating polypeptide is a phosphatase.

28. The sample analysis cartridge according to claim 26, characterized in that: The hydrolase is selected from the group consisting of β-galactosidase, β-glucuronidase and β-glucosidase.

29. The sample analysis cartridge according to claim 1 or 2, characterized in that: The electroactive analyte is chlorophenol red-β-D-pyranoside (CPRG), and the activating compound is β-galactosidase, and when the electroactive analyte contacts the activating compound, chlorophenol red (CPR) is formed; or, the electroactive analyte is p-nitrophenol-β-D-glucuronide (PNPG), and the activating compound is β-glucuronidase, and when the electroactive analyte contacts the activating compound, p-nitrophenol (PNP) is formed; or, the electroactive analyte is p-diphenol-β-D-pyranoside (PDPG), and the The activating compound is β-glucosidase, and when the electroactive analyte contacts the activating compound, p-diphenol (PDP) is formed; the electroactive analyte is p-aminophenol-β-pyranogalactoside (PAPG), and the activating compound is β-galactosidase, and when the electroactive analyte contacts the activating compound, p-aminophenol (PAP) is formed; or, the electroactive analyte is p-aminophenyl phosphate (PAPP), and the activating compound is a phosphatase, and when the electroactive analyte contacts the activating compound, p-aminophenol (PAP) is formed.

30. The sample analysis cartridge according to claim 1 or 2, characterized in that: The fluid parameter being detected is a physical property.

31. The sample analysis cartridge according to claim 1 or 2, characterized in that: The fluid parameter being detected is a chemical substance.

32. The sample analysis cartridge according to claim 31, characterized in that: The chemical substance is an organic chemical compound.

33. The sample analysis cartridge according to claim 31, characterized in that: The chemical substances are inorganic chemical compounds.

34. The sample analysis cartridge according to claim 31, characterized in that: The fluid parameter being detected is a toxic chemical substance.

35. A method for voltammetric detection of fluid parameters in a fluid, characterized in that: The method comprises: providing a fluid sample including a fluid parameter, the fluid sample being releasably collected in a fluid collection device; Inserting the end of the fluid collection device into a cartridge comprising: at least one sample analysis reservoir operable to receive the end of a fluid collection device, the at least one sample analysis reservoir comprising: a first compartment, the first compartment containing an activation compound or an electroactive analyte, the electroactive analyte being activated by the activation compound to form an activated electroactive analyte when the activation compound is activated by the fluid parameter in the fluid sample, the first compartment having an opening at an upper end; a second compartment, the second compartment comprising the electroactive analyte, the electroactive analyte being activated by the activating compound to form the activated electroactive analyte when the first compartment comprises the activating compound, or the second compartment comprising the activating compound when the first compartment comprises the electroactive analyte; and a penetrable barrier disposed between the first compartment and the second compartment to fluidly separate the first compartment and the second compartment and further configured to be engageable by the fluid collection device; and a voltammetric sensor disposed at least partially within the second compartment; moving the end of the fluid collection device into the first compartment and penetrating the barrier with the end of the fluid collection device to fluidly connect the first compartment and the second compartment; releasing the fluid sample from the fluid collection device into the at least one sample analysis reservoir, causing the fluid parameters to activate the activating compound to produce the activated activating compound, which then contacts the electroactive analyte to form the activated electroactive analyte, which then contacts the voltammetric sensor; applying a voltage to the voltammetric sensor; detecting a current passing through the volt-ampere sensor; and The detected current is compared to a threshold value to determine the presence of the fluid parameter in the fluid sample.

36. The method for voltammetric detection of fluid parameters in a fluid according to claim 35, characterized in that: When the first compartment contains the activating compound, the fluid sample is released in the first compartment before penetrating the barrier.

37. The method for voltammetric detection of fluid parameters in a fluid according to claim 35, characterized in that: When the second compartment contains the activating compound, the fluid sample is released in the second compartment after penetrating the barrier.

38. The method for voltammetric detection of fluid parameters in a fluid according to any one of claims 35 to 37, characterized in that: The first compartment includes a second penetrable barrier disposed over the opening, and the method includes moving the fluid collection device with sufficient force to penetrate the second penetrable barrier and move into the first compartment.

39. A method for manufacturing a sample analysis cartridge for voltammetric detection of fluid parameters in a fluid sample, characterized in that: The method comprises: One or more sample storages are formed for the sample analysis cartridge, wherein each sample storage is made as follows: forming a bottom cartridge housing portion having a volt-ampere sensor therein; placing an electroactive analyte or activating compound in the bottom cartridge housing portion; forming a top cartridge housing portion; forming a penetrable barrier to separate the bottom cartridge housing portion and the top cartridge housing portion; placing an activating compound in the top cartridge housing portion when the electroactive analyte is placed in the bottom cartridge housing portion, or placing the electroactive analyte in the top cartridge housing portion when the activating compound is placed in the bottom cartridge housing portion; and (a) placing the penetrable barrier on top of the bottom cartridge housing portion; and slidably coupling the bottom cartridge housing portion and the top cartridge housing portion to form a cartridge; or (b) slidably coupling the bottom cartridge housing portion and the top cartridge housing portion to form a cartridge, the cartridge comprising a slot between the bottom cartridge housing portion and the top cartridge housing portion to slidably receive the penetrable barrier; and The penetrable barrier is slidably inserted into the receptacle, wherein the penetrable barrier is configured to be engageable by an external fluid collection device that provides the fluid sample.

40. The method for manufacturing a sample analysis cartridge according to claim 39, characterized in that: The method also includes, after slidably coupling the bottom cartridge housing portion and the top cartridge housing portion, providing a second penetrable barrier disposed over an opening of the top cartridge housing portion.

41. The method for manufacturing a sample analysis cartridge according to claim 39 or 40, characterized in that: The permeable barrier is formed to include one or more recesses to contain the activating compound or the electroactive analyte.

42. The method for manufacturing a sample analysis cartridge according to claim 39 or 40, characterized in that: The bottom housing compartment is formed to include one or more recesses to contain the electroactive analyte or the activating compound.

43. The method for manufacturing a sample analysis cartridge according to claim 39, wherein: The penetrable barrier is formed of a material that can be torn by the end of the fluid collection device.

44. The method for manufacturing a sample analysis cartridge according to claim 39, wherein: The method includes forming two, three, four, five or six sample reservoirs for the cartridge.

45. A voltammetric detection device for detecting fluid parameters in a fluid sample contained in a sample analysis cartridge, characterized in that: The volt-ampere detection device comprises: at least one slot for releasably inserting the sample analysis cartridge as defined in any one of claims 1 to 34; a voltage source configured to apply a voltage to a voltammetric sensor in a reservoir of the sample analysis cartridge; a current detector for detecting a current passing through the sensor when the voltage is applied to the volt-ampere sensor; and A controller is operably coupled to the voltage source and the current detector and is configured to control the operation of the volt-ampere detection device.

46. ​​The volt-ampere detection device according to claim 45, characterized in that: The volt-ampere detection device includes two, three, four, five or six slots.

47. The volt-ampere detection device according to claim 45 or claim 46, characterized in that: The voltage detector is operably coupled to the memory device.

48. A volt-ampere detection assembly, characterized in that: The components include: A voltammetric detection device, the voltammetric detection device is used to detect fluid parameters in a fluid sample, the voltammetric detection device comprises: at least one slot for releasably inserting a sample analysis cartridge as defined in any one of claims 1 to 34; a voltage source configured to apply a voltage to a voltammetric sensor in a reservoir of the sample analysis cartridge; a current detector for detecting a current through the voltammetric sensor when the voltage is applied to the sensor; and a controller operably coupled to the voltage source and the current detector and configured to control operation of the volt-ampere detection device; and A fluid collection device is provided for releasably collecting a fluid sample for insertion into the sample analysis cartridge.

49. Use of a sample analysis cartridge according to any one of claims 1 to 34 for voltammetric detection of a fluid parameter in a fluid sample.

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