Devices, systems, and methods for measuring solution characteristic of sample comprising microorganisms

Abstract

To provide various apparatuses, systems, and methods for measuring a solution characteristic of a sample comprising microorganisms.SOLUTION: In one embodiment, a sensor apparatus is disclosed comprising a sample container comprising a sample chamber configured to receive a sample and a reference sensor component comprising a reference conduit having a reference conduit cavity defined therein. The reference conduit cavity can be at least partially filled with a reference buffer gel, buffer solution, or wicking component. A segment of the reference conduit can extend into the sample chamber. A reference electrode material can be positioned at a proximal end of the wicking component or extend partially into the reference conduit cavity. The sensor apparatus can also comprise an active sensor component having an active electrode in fluid contact with the sample. The sample in the sample chamber can be aerated through an aeration port defined along a surface of the sample container.SELECTED DRAWING: Figure 9A

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 687,167, filed June 19, 2018, which is incorporated by reference in its entirety. Technical Field

[0002] The present disclosure relates generally to diagnostic devices for measuring solution properties of a sample, and more particularly to devices, systems and methods for measuring solution properties of a sample containing a microorganism. background

[0003] Infections caused by anti-infective resistant infectious agents or microorganisms are a significant problem for medical professionals in hospitals, nursing homes, and other medical environments. For example, such infections can lead to a potentially life-threatening complication known as sepsis, in which chemicals released into the bloodstream by the infectious agent can trigger a critical systemic inflammatory response as well as a vasoactive response that can cause fever, hypotension, and death. When faced with such infections, the preferred course of action is for clinicians to use anti-infective compounds judiciously, preferably using only those necessary to mitigate the infection.

[0004] However, the most common practice today is to administer broad-spectrum anti-infective drugs, often multiple drugs, to patients to ensure the adequacy of treatment until the organism is identified and tested for drug susceptibility. This tends to result in multi-drug resistant infectious agents. Ideally, the susceptibility of an infectious agent would be detected immediately after its presence is confirmed. To determine the susceptibility of such infectious agents to anti-infective drugs, samples containing the infectious agent must be quantified, which requires analyzing the sample for microbial growth or lack thereof.

[0005] Existing biosensors used to analyze infectious agents in biological or other types of samples include an active sensor element having an active electrode and a reference sensor element having a reference electrode immersed in a reference solution in ion exchange contact with the sample of interest. Traditionally, this is done using an Ag / AgCl electrode in an aqueous reference buffer, such as KCl. However, this traditional sensor design is not ideal for a variety of reasons. First, silver or silver chloride can diffuse into the sample and inhibit the growth of microorganisms in the sample, thus complicating any measurements performed on the sample. Furthermore, traditional reference solutions are often provided in disposable containers with thin glass walls, which are expensive to manufacture, store, and transport. Furthermore, diffusion of microorganisms through salt bridges formed between the sample and the reference solution can contaminate the reference solution. Moreover, traditional biosensors often require the reference electrode to be located on the silicon die surface, which uses valuable wafer real estate, further increasing the cost of such sensors.

[0006] As a result of the above limitations and restrictions, there is a need for improved devices, systems and methods for rapidly and effectively assaying samples containing microorganisms for microbial growth or lack thereof. Such solutions should be cost-effective and address the shortcomings of conventional biosensors. Summary of the Invention

[0007] Disclosed are devices, systems and methods for measuring solution properties (e.g., ORP or pH) of a sample containing microorganisms. In one embodiment, a sensor device for measuring solution properties of a sample is disclosed. The sensor device can include a sample container including a sample chamber configured to receive a sample and a reference sensor member. The reference sensor member can include a reference conduit including a reference conduit cavity, a reference conduit first opening and a reference conduit second opening. The reference sensor member can also include a wicking member extending through the reference conduit cavity having a wick distal end and a wick proximal end. At least a portion of the wicking member can be in fluid communication with the sample chamber such that at least a portion of the sample is drawn by the wicking member toward the wick proximal end. The reference sensor member can also include a reference electrode material disposed at the wick proximal end.

[0008] The sensor apparatus may further comprise an active sensor member including an active electrode material, at least a portion of which extends into the sample chamber and is capable of fluid contact with the sample when the sample chamber is at least partially filled with the sample.

[0009] The reference and active sensor members can be electrically coupled to a parameter analyzer by conductive connections, and solution properties of the sample can be determined based on the potential difference measured between the active and reference electrode materials.

[0010] The sensor device may include a container cap configured to be removably coupled to the sample container. The reference conduit may extend from a bottom surface of the container cap. The container cap may be made in part of a non-conductive material. The container cap may be made in part of a transparent non-conductive material such that at least a portion of the wicking member is visible through the container cap.

[0011] The sample reservoir can be made in part of at least one of a ceramic material and a polymeric material. The reference electrode material can be a conductive ink that is provided or dispensed on the proximal end of the wick. The conductive ink provided or dispensed on the proximal end of the wick can be hardened by curing. In some embodiments, the conductive ink can be a silver-silver chloride ink.

[0012] The wicking member can be made in part of a porous polymeric material. In some embodiments, the wicking member can be made in part of high density polyethylene (HDPE). In other embodiments, the wicking member can be made in part of natural fibers. The wicking member can include pores sized between about 15 μm and about 150 μm.

[0013] The wicking member may be treated with a surfactant such that at least a surface of the wicking member is coated with the surfactant. The surfactant may be configured to increase the hydrophilicity of the wicking member.

[0014] The sensor device may further include a vent defined along at least one of a bottom surface and a side surface of the sample container. In some embodiments, a hydrophobic breathable membrane may cover the vent. The vent and hydrophobic breathable membrane may be configured such that air may enter the sample chamber through the vent and the hydrophobic breathable membrane to aerate the sample chamber.

[0015] When the reference sensor member is implemented as a container cap, the container cap can be configured to be removably coupled to the sample container via the attachment connection, and the additional hydrophobic breathable membrane can cover at least a portion of the bottom surface of the container cap. In these embodiments, when the container cap is screwed onto the sample container via the attachment connection (e.g., a screw connection), an airflow path can be established. The airflow path can include an air vent, a hydrophobic breathable membrane, a sample chamber, an additional hydrophobic breathable membrane, and an air gap defined between the container cap and the sample container along the attachment connection.

[0016] In some embodiments, the solution property measured by the sensor device is pH, and the active electrode material of the active sensor component can be made in part of a pH-sensitive material, hi some embodiments, the pH-sensitive material includes at least one of silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium dioxide, ruthenium dioxide, and zirconium dioxide.

[0017] In other embodiments, the solution property measured can be the oxidation-reduction potential (ORP) of the sample and the active electrode material of the active sensor element can be made in part of a redox-sensitive material. The redox-sensitive material can include at least one of platinum, gold, silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium dioxide, ruthenium dioxide, and zirconium dioxide. In these and other embodiments, the parameter analyzer can be or comprise at least a portion of a voltmeter and a multimeter.

[0018] A reference sensor member is also disclosed. The reference sensor member can be configured to removably couple to a sample vessel configured to receive a sample. The reference sensor member can also include a reference conduit including a reference conduit cavity. In some embodiments, the reference sensor member can be implemented as a vessel cap. The reference conduit can extend from an underside of the vessel cap.

[0019] The reference sensor member may comprise a wicking member disposed within a portion of the reference conduit cavity having a wick distal end and a wick proximal end. At least a portion of the wicking member may be configured to be in fluid contact with the sample upon attachment of the container cap to the sample container such that at least a portion of the sample is drawn by the wicking member toward the wick proximal end. The reference sensor member may also comprise a reference electrode material disposed at the wick distal end. The reference electrode material may be configured to exhibit a substantially stable electrode potential relative to an active electrode in electrical communication with the reference electrode material.

[0020] The reference sensor member may be made in part from a non-conductive material. If the reference sensor member is implemented as a container cap, the container cap may be made in part from a non-conductive material. In some embodiments, the container cap may be made in part from a transparent non-conductive material such that at least a portion of the wicking member is visible through the container cap.

[0021] In some embodiments, the reference electrode material can be a conductive ink provided or dispensed on the distal end of the wick. The conductive ink provided or dispensed on the proximal end of the wick can be hardened by curing. In some embodiments, the conductive ink can be a silver-silver chloride ink.

[0022] The wicking member may be made in part from a porous polymeric material. In another embodiment, the wicking member may be made in part from natural fibers. The wicking member may include pores sized from about 15 μm to about 150 μm.

[0023] The wicking member may be treated with a surfactant such that at least a surface of the wicking member is coated with the surfactant. The surfactant may be configured to increase the hydrophobicity of the wicking member.

[0024] Also disclosed is a method of measuring solution properties of a sample, which may include filling a sample chamber of a sample container with a sample containing an infectious pathogen, attaching a reference sensor member (e.g., implemented as a container cap) to the sample container, electrically coupling a reference electrode material of the reference sensor member to a parameter analyzer, and electrically coupling the parameter analyzer to an active sensor including an active electrode material, and measuring solution properties of the sample based on a potential difference measured between the active electrode material and the reference electrode material.

[0025] At least a portion of the active electrode material extends into the sample chamber and is in fluid contact with the sample. The reference sensor member can include a reference conduit having a reference conduit cavity, a reference conduit first opening and a reference conduit second opening. The reference conduit cavity can be at least partially filled with a wicking member extending through the reference conduit cavity having a wick distal end and a wick proximal end.

[0026] At least a portion of the wicking member may be in fluid contact with the sample in the sample chamber, and at least a portion of the sample may be drawn by the wicking member toward the proximal end of the wick (e.g., by capillary action).

[0027] The reference electrode material can be disposed at the proximal end of the wick. The reference electrode material can be a conductive ink provided or dispensed on the proximal end of the wick. The conductive ink can be hardened by curing. In some embodiments, the conductive ink can be a silver-silver chloride ink.

[0028] The method may further include pumping air into the sample chamber through an air vent defined along at least one of a bottom and side surface of the sample container and through a hydrophobic breathable membrane covering the air vent, wherein the air pumped into the sample chamber aerates the sample. The air pumped into the sample chamber may exit the sample chamber through an additional breathable membrane covering at least a portion of a lower surface of the container cap and through an air gap defined between the container cap and the sample container along the attachment connection.

[0029] Determining the solution properties of the sample can include measuring the pH of the sample. The active electrode material can be made of a portion of a pH-sensitive material. In some embodiments, the pH-sensitive material can include at least one of silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium dioxide, ruthenium dioxide, and zirconium dioxide.

[0030] In other embodiments, determining the solution properties of the sample includes measuring the oxidation-reduction potential (ORP) of the sample, where the active electrode material is made in part of a redox-sensitive material that may include at least one of platinum, gold, silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium dioxide, ruthenium dioxide, silicon dioxide, and zirconium dioxide.

[0031] A method of manufacturing a reference sensor member is disclosed. The method includes providing a container cap configured to be removably coupled to a sample container configured to receive a sample. A reference conduit extends from a bottom surface of the container cap. The reference conduit may include a reference conduit cavity. The method may also include disposing a wicking member in the reference conduit cavity. The wicking member may include a wick distal end and a wick proximal end. The method may further include providing or dispensing a conductive ink onto the wick proximal end and curing the conductive ink until the conductive ink is hard.

[0032] The method may further include providing or dispensing a volume of the conductive ink between about 50 μL and 500 μL onto the wick proximal end. The conductive ink may be cured at a temperature above 100° C. The conductive ink may be cured for a time period between about 60 minutes and about 180 minutes.

[0033] In some embodiments, the reference conduit may be gradually tapered. The wicking member may be shaped such that it gradually tapers from the wick proximal end to the wick distal end.

[0034] Another embodiment of a sensor apparatus for measuring the pH of a sample is disclosed. The sensor apparatus may include a sample vessel including a sample chamber configured to receive a sample and a reference sensor member.

[0035] The reference sensor member comprises a reference conduit having a reference cavity, a reference conduit first opening and a reference conduit second opening. In some embodiments, the reference conduit cavity can be partially filled with a reference buffer gel. A segment of the reference conduit can extend into the sample chamber. The reference conduit second opening can be configured to allow the sample to be in fluid contact with the reference buffer gel. The reference electrode can extend partially into the reference conduit filled with the reference buffer gel.

[0036] The sensor device may further comprise an active sensor member comprising an active electrode extending into the sample chamber and partially contained within the active electrode housing. The active electrode may include a sample contacting surface.

[0037] The active electrode can be disposed at a distal end of an active electrode housing, and the active electrode housing can be configured to expose the sample contacting surface such that the sample contacting surface is in fluid communication with the sample.

[0038] The conductive contact layer can be coupled to the active electrode and disposed proximate to the active electrode within the active electrode housing. The conductive contact layer and the reference electrode can be electrically coupled by a conductive connection to a voltmeter. The pH of the sample can be measured based on the potential difference between the active electrode and the reference electrode.

[0039] The reference conduit may have a conduit wall that may gradually taper from the reference conduit proximal end to the reference conduit distal end such that a first open area of ​​the reference conduit first opening is greater than a second open area of ​​the reference conduit second opening.

[0040] The reference electrode may include a reference electrode tip. The reference electrode tip may be positioned proximate to the second opening of the reference conduit such that when the reference electrode extends partially into the reference conduit cavity, a volume of reference buffer gel separates the reference electrode tip from the second opening of the reference conduit. The reference conduit cavity may be filled with about 100 μL to about 500 μL of reference buffer gel.

[0041] The reference electrode can be made in part of one or more metals, hi some embodiments, the reference electrode can be made in part of platinum, stainless steel, or a combination thereof.

[0042] The active electrode can be made in part of a metal oxide, hi some embodiments, the active electrode can be made in part of silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium oxide, ruthenium dioxide, zirconium dioxide, or combinations thereof.

[0043] The conductive contact layer may be made in part of one or more metals. The conductive contact layer may be made in part of aluminum, copper, platinum, or a combination thereof.

[0044] In some embodiments, the reference buffer gel can be an agar gel formed from agar powder and a reference buffer. The agar powder in the agar gel can have a concentration of about 1% (w / v%) to about 5% (w / v%).

[0045] The reference buffer may include potassium ferricyanide (III), potassium ferricyanide (II), potassium dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof, hi another embodiment, the reference buffer may include potassium chloride.

[0046] The sensor device may include a vent conduit. A segment of the vent conduit may extend into the sample chamber. The vent conduit may be configured to aerate the sample in the sample container.

[0047] Another embodiment of a sensor apparatus for measuring the oxidation-reduction potential (ORP) of a sample is also disclosed. The sensor apparatus may include a sample container including a sample chamber configured to receive a sample and a reference sensor member.

[0048] The reference sensor member can include a reference conduit having a reference conduit cavity, a reference conduit first opening, and a reference conduit second opening. The reference conduit cavity can be at least partially filled with a reference buffer gel. A segment of the reference conduit can extend into the sample chamber.

[0049] The second opening of the reference conduit can be configured to allow the sample to be in fluid contact with the reference buffer gel, and the reference electrode can extend partially into the reference conduit cavity filled with the reference buffer gel.

[0050] The sensor device may further comprise an active sensor element. The active sensor element may further comprise an active electrode housing extending into the sample chamber and an active electrode partially contained within the active electrode housing. The active electrode may have a sample contacting surface. The active electrode may be disposed at a distal end of the active electrode housing, and the active electrode housing may be configured such that the sample contacting surface is exposed such that the sample contacting surface is in fluid communication with the sample. The active electrode and the reference electrode may be electrically coupled to a voltmeter by a conductive connection. The ORP of the sample may be measured based on a potential difference between the active electrode and the reference electrode.

[0051] The reference conduit may include a conduit wall. The conduit wall may be gradually tapered from the reference conduit proximal end to the reference conduit distal end such that a first opening area of ​​the reference conduit first opening is greater than a second opening area of ​​the reference conduit second opening. The reference conduit cavity may be filled with about 100 μL to about 500 μL of reference buffer gel.

[0052] The reference electrode can include a reference electrode tip, which can be positioned proximate to the reference conduit cavity such that when the reference electrode extends partially into the reference conduit cavity, a volume of reference buffer gel separates the reference electrode tip from the second opening of the reference conduit.

[0053] The reference electrode can be made in part from one or more metals, hi some embodiments, the reference electrode can be made in part from platinum, stainless steel, or a combination thereof.

[0054] The active electrode can be made in part of one or more metals, one or more metal oxides, or a combination thereof For example, the active electrode can be made in part of platinum, gold, silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium dioxide, ruthenium dioxide, zirconium dioxide, or a combination thereof.

[0055] The reference buffer gel can be an agar gel formed from agar powder and the reference buffer gel. The agar powder in the agar gel can have a concentration of about 1% (w / v%) to about 5% (w / v%).

[0056] The reference buffer may include potassium ferricyanide (III), potassium ferricyanide (II), potassium dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof, hi another embodiment, the reference buffer may include potassium chloride.

[0057] The sensor device may include a vent conduit or vent. A segment of the vent conduit may extend into the sample chamber. The vent conduit may be configured to aerate the sample in the sample container.

[0058] Another embodiment of a sensor apparatus for measuring the pH of a sample is also disclosed. The sensor apparatus can include a sample container including a sample chamber configured to receive a sample and a reference sensor member.

[0059] The reference sensor member may comprise a reference conduit including a reference conduit proximal end defining a reference conduit first opening, a reference conduit distal end defining a reference conduit second opening, and a reference conduit cavity between the reference conduit proximal end and the reference conduit distal end.

[0060] The reference conduit cavity can be at least partially filled with a reference buffer. A segment of the reference conduit can extend into the sample chamber. The reference electrode can extend partially into the reference conduit cavity filled with the reference buffer.

[0061] An ion exchange membrane can be coupled to the distal end of the reference conduit and configured to block the second opening of the reference conduit. The ion exchange membrane can be configured to prevent mixing of the sample with the reference buffer but allow ions to pass through the ion exchange membrane.

[0062] The sensor device may further comprise an active sensor member, the active sensor member further comprising an active electrode housing extending into the sample chamber and an active electrode partially contained within the active electrode housing.

[0063] The active electrode can have a sample contacting surface. The active electrode can be disposed at a distal end of an active electrode housing, and the active electrode housing can be configured to expose the sample contacting surface such that the sample contacting surface is in fluid communication with the sample.

[0064] The conductive contact layer can be coupled to the active electrode within the active electrode housing and can be positioned proximate to the active electrode. The conductive contact layer and the reference electrode can be electrically coupled by a conductive connection to a voltmeter, where the pH of the sample is measured based on the potential difference between the active electrode and the reference electrode.

[0065] The reference conduit may include a conduit wall. The conduit wall may be gradually tapered from the reference conduit proximal end to the reference conduit distal end such that the reference conduit first opening is larger than the reference conduit second opening. The reference conduit cavity may be filled with about 100 μL to about 500 μL of the reference buffer.

[0066] The reference electrode can include a reference electrode tip. When the reference electrode extends partially into the reference conduit cavity such that a volume of reference buffer separates the reference electrode tip from the ion exchange membrane, the reference electrode tip can be proximal to the ion exchange membrane.

[0067] In some embodiments, the ion exchange membrane can be a perfluorosulfonated ionomer membrane. For example, the perfluorosulfonated ionomer membrane can be a Nafion® membrane. The ion exchange membrane can have a thickness of about 25 μm to about 180 μm.

[0068] The reference electrode can be made in part of one or more metals, for example, the reference electrode can be made in part of platinum, stainless steel, or a combination thereof.

[0069] The active electrode can be made in part of a metal oxide, for example, the active electrode can be made in part of silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium dioxide, ruthenium dioxide, zirconium dioxide, or combinations thereof.

[0070] The conductive contact layer may be made in part of one or more metals, hi some embodiments, the conductive contact layer may be made in part of aluminum, copper, platinum, or combinations thereof.

[0071] The reference buffer can include potassium ferricyanide (III), potassium ferricyanide (II), potassium dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof, hi other embodiments, the reference buffer can include potassium chloride.

[0072] The sensor apparatus may further comprise a vent conduit, a segment of the vent conduit extending into the sample chamber, where the vent conduit is configured to aerate the sample in the sample container.

[0073] The reference sensor member may be removably coupled to the sample vessel such that the reference sensor member is detachable from the sample vessel.

[0074] Another embodiment of a sensor apparatus for measuring an oxidation-reduction potential (ORP) of a sample is disclosed, the sensor apparatus comprising a sample container including a sample chamber configured to receive a sample, and a reference sensor member.

[0075] The reference sensor member may comprise a reference conduit including a reference conduit proximal end defining a reference conduit first opening, a reference conduit distal end defining a reference conduit second opening, and a reference conduit cavity between the reference conduit proximal end and the reference conduit distal end. The reference conduit cavity may be at least partially filled with a reference buffer. A segment of the reference conduit may extend into the sample chamber. The sensor device may further comprise a reference electrode extending partially into the reference conduit cavity filled with the reference buffer.

[0076] The ion exchange membrane can be coupled to the distal end of the reference conduit and can be configured to block the second opening of the reference conduit. The ion exchange membrane can be configured to prevent mixing of the sample with the reference buffer but allow ions to cross (pass) through the ion exchange membrane.

[0077] Additionally, the sensor device may include an active sensor element that may include an active electrode housing extending into the sample chamber and an active electrode partially contained within the active electrode housing.

[0078] The active electrode can have a sample contacting surface. The active electrode can be disposed at a distal end of an active electrode housing, and the active electrode housing is configured to expose the sample contacting surface such that the sample contacting surface is in fluid communication with the sample.

[0079] The active electrode and the reference electrode can be electrically coupled to a voltmeter by a conductive connection, and the ORP of the sample can be measured based on the potential difference between the active electrode and the reference electrode.

[0080] The reference conduit may include a conduit wall. In some embodiments, the conduit wall may be gradually tapered from the reference conduit proximal end to the reference conduit distal end such that a first opening area of ​​the reference conduit first opening is greater than a second opening area of ​​the reference conduit second opening. The reference conduit cavity may be filled with about 100 μL to about 500 μL of the reference buffer solution.

[0081] The reference electrode can comprise a reference electrode tip, which can be positioned proximal to the ion exchange membrane when the reference electrode extends partially into the reference conduit cavity such that a volume of reference buffer separates the reference electrode tip from the ion exchange membrane.

[0082] The reference electrode can be made in part of one or more metals, for example, the reference electrode can be made in part of platinum, stainless steel, or a combination thereof.

[0083] The active electrode can be made in part of one or more metals, one or more metal oxides, or a combination thereof For example, the active electrode can be made of platinum, gold, silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium dioxide, ruthenium dioxide, zirconium dioxide, or a combination thereof.

[0084] In some embodiments, the reference buffer may include potassium ferricyanide (III), potassium ferricyanide (II), potassium dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof, hi other embodiments, the reference buffer may include potassium chloride.

[0085] As previously described, the ion exchange membrane can be coupled to the distal end of the reference conduit and configured to block the second opening of the reference conduit. In some embodiments, the ion exchange membrane can be a perfluorosulfonated ionomer membrane. For example, the perfluorosulfonated ionomer membrane can be a Nafion® membrane. The ion exchange membrane can have a thickness of about 25 μm to about 180 μm.

[0086] The sensor apparatus may further comprise a vent conduit. A segment of the vent conduit may extend into the sample chamber. The vent conduit may be configured to aerate the sample in the sample container.

[0087] The reference sensor member may be removably coupled to the sample vessel such that the reference sensor member is detachable from the sample vessel. [Brief description of the drawings]

[0088] [Figure 1A] FIG. 1A illustrates one embodiment of a sensor apparatus for measuring the pH of a sample.

[0089] [Figure 1B] FIG. 1B illustrates one embodiment of a sensor apparatus for measuring the ORP of a sample.

[0090] [Figure 1C] FIG. 1C illustrates another embodiment of a sensor apparatus for measuring the pH of a sample.

[0091] [Figure 1D] FIG. 1D illustrates another embodiment of a sensor apparatus for measuring the ORP of a sample.

[0092] [Diagram 2] FIG. 2 illustrates a top perspective view of one embodiment of a sensor device.

[0093] [Figure 3A] FIG. 3A shows a vertical cross-sectional view of one embodiment of a reference sensor member.

[0094] [Figure 3B] FIG. 3B shows a vertical cross-sectional view of another embodiment of a reference sensor member.

[0095] [Figure 4]FIG. 4 shows a schematic diagram of the active and reference electrodes of the sensor device connected to a high impedance voltmeter.

[0096] [Diagram 5] FIG. 5A depicts ORP measurements made with a conventional KCl reference solution and a stainless steel reference electrode in a reference buffer gel after addition of a high potentiometric ORP solution.

[0097] FIG. 5B represents pH measurements made with a conventional KCl reference solution and a platinum electrode in the reference buffer gel after exchanging the pH 7 solution with a pH 4 solution.

[0098] [Figure 6] FIG. 6 shows ORP measurements of E. coli bacterial growth using a reference electrode in 1% agar gel buffer, 5% agar gel buffer, and conventional KCl reference solution.

[0099] [Figure 7] FIG. 7A depicts several ORP measurements taken when the conventional ORP buffer was replaced with a high potential ORP solution and then switched back to the conventional ORP buffer.

[0100] FIG. 7B represents the ORP measurement of E. coli bacterial growth using a conventional ORP probe and the potential difference between a reference sensor element having an ion exchange membrane and a KCl reference solution during the measurement period.

[0101] [Figure 8] FIG. 8 shows another embodiment of a sensor apparatus for measuring solution properties of a sample.

[0102] [Figure 9A] FIG. 9A shows a vertical cross-sectional view of another embodiment of a sensor device.

[0103] [Figure 9B] FIG. 9B illustrates one embodiment of a reference sensor member.

[0104] [Figure 10] FIG. 10 shows the change in oxidation-reduction potential (ORP) of three sample aliquots measured over time using three different sensor settings.

[0105] [Figure 11] FIG. 11 shows the results of cell counts performed on smeared bacterial colonies of output samples containing different strains of E. coli.

[0106] [Figure 12] FIG. 12 shows the effect of aeration on the growth rate of two samples containing Pseudomonas aeruginosa.

[0107] [Figure 13] FIG. 13 shows the effect of aeration on the preparation time of two 0.5 McFarland bacterial samples using the sensor device. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0108] Detailed Description The variations of the devices, systems and methods described herein are best understood from the detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings may not be to scale. Dimensions of certain features have been expanded or reduced for clarity, and not all features may be visible or labeled in every drawing. The drawings are not intended to limit or restrict the scope of the claims to what is illustrated.

[0109] Figure 1A illustrates one embodiment of a sensor apparatus 100 for measuring the pH of a sample 102. Figure 1A illustrates a vertical cross-sectional view of the sensor apparatus 100.

[0110] The sensor device 100 may include a sample container 104 having a sample chamber 106 configured to receive a sample 102. The sample 102 may include at least one of a sample obtained from a patient or subject, a biological sample, an environmental sample, and a food sample. The sample 102 obtained from a patient or subject may include at least one of a bodily fluid of the patient or subject and a swab obtained from the patient or subject.

[0111] In some embodiments, the patient or subject can be a human patient or subject, hi other embodiments, the patient or subject can be a non-human animal patient or subject.

[0112] In some embodiments, the bodily fluid can include blood, urine, serum, plasma, saliva, sputum, semen, breast milk, joint fluid, spinal fluid such as cerebrospinal fluid, wound material, mucus, stool-associated fluid, vaginal secretions, synovial fluid, sputum, peritoneal fluid, pericardial fluid, amniotic fluid, or combinations thereof.

[0113] In these and other embodiments, the specimen (swab) obtained from the patient or subject can include a wound swab, a rectal swab, a vaginal swab, a resuspension of the aforementioned swabs, or a combination thereof.

[0114] In all such embodiments, the sample 102 may contain multiple microorganisms or infectious agents. The disclosed devices, systems and methods may be used to assay the sample 102 for microbial growth as part of a microbiological quantification or antibiotic susceptibility testing (AST) method.

[0115] In some embodiments, sample 102 can include or refer to a bacterial culture derived from at least one of a sample obtained from a patient or subject, a biological sample, an environmental sample, and a food sample. For example, sample 102 can include or refer to a bacterial culture or resuspended bacterial culture derived from a bodily fluid or specimen obtained from a patient or subject. As a more specific example, sample 102 can include a bacterial culture or resuspended bacterial culture derived from a bodily fluid or swab obtained from a patient or subject that tests positive for microbial growth.

[0116] More specifically, the sample 102 can include a bacterial culture derived from blood obtained from a patient or subject that tests positive for microbial growth. In some embodiments, the sample 102 is or refers to a positive blood culture. For purposes of this disclosure, a positive blood culture can be a bacterial culture derived from blood taken from a patient or subject that tests positive for bacterial growth. For example, a patient may exhibit symptoms of sepsis (e.g., high fever, chills, etc.), and blood (e.g., 5 mL to 10 mL) can be drawn from the patient and transferred into a commercially available blood culture container or tube containing bacterial growth medium (e.g., 30 mL to 40 mL of growth medium). The blood culture container or tube can then be incubated at 35° C.±2° C. to allow bacteria to grow. If the patient's blood is contaminated with bacteria, the bacteria will replicate within the container or tube. A blood culture system or device can then be used to monitor bacterial growth (such as by monitoring bacterial CO2 production in a vessel or tube), and the system or device can determine that the sample tests "positive" for bacterial growth if a critical CO2 threshold is met. Depending on the type of pathogen and growth rate, blood cultures can be positive for anywhere from 7 hours to 3 days. Such "positive blood cultures" can then be used for further downstream testing, such as using any of the devices, systems and methods disclosed herein.

[0117] In additional embodiments, the sample 102 may include an environmental sample obtained from a water stream, river, lake, ocean, contaminated area, quarantine zone, emergency area, or combinations thereof. In other embodiments, the sample 102 may include a food sample obtained from a food preparation facility, a food facility, a waste facility, or combinations thereof.

[0118] In some embodiments, an aqueous growth medium may be added to the sample 102 prior to introduction into the sample vessel 104. In other embodiments, an aqueous growth medium may be added to the sample 102 once the sample 102 has been injected, delivered, or otherwise introduced into the sample vessel 104.

[0119] In one embodiment, the aqueous growth medium can be Mueller-Hinton Broth (MHG) supplemented with glucose, in another embodiment, the aqueous growth medium can be a solution containing bactotryptone, tryptic soy digest, yeast extract, beef extract, cation-adjusted Mueller-Hinton Broth (CAMHB), starch, acid hydrolysate of casein, calcium chloride, magnesium chloride, sodium chloride, blood or hemolyzed blood such as horse hemolyzed blood (LHB), CAMHB-LHB, glucose or other carbohydrates, or a combination thereof.

[0120] Microorganisms or infectious agents that can be assayed using the devices, methods, and systems disclosed herein can be any metabolic unicellular or multicellular organism, including bacteria and fungi. In some embodiments, the microorganism or infectious agent is selected from the group consisting of Acinetobacter, Acetobacter, Actinomyces, Aerococcus, Aeromonas, Agrobacterium, Anaplasma, Azorhizobium, Azotobacter, Bacillus, and the like. Bacillus, Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Chlamydia, Chlamydophila a), Citrobacter, Clostridium, Corynebacterium, Coxiella, Ehrlichia, Enterobacter, Enterococcus, Escherichia, Francisella, Fusobacterium ), Gardnerella, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Legionella, Listeria, Methanobacterium, Microbacterium, Micrococcus,Morganella, Moraxella, Mycobacterium, Mycoplasma, Neiserria, Pandoraea, Pasteurella, Pestostreptococcus, Porphyromonas, Prevotella, Proteus, Providencia, Pseudomonas, Ralstonia, Raoutella, Rhizobium, Rickettsia The microorganism or infectious agent may be a bacterium selected from the genera Ickettsia, Rochalimaea, Rothia, Salmonella, Serratia, Shewanella, Shigella, Spirillum, Staphylococcus, Strenotrophomonas, Streptococcus, Streptomyces, Treponema, Vibrio, Wolbachia, Yersinia, or a combination thereof. In other embodiments, the microorganism or infectious agent may be one or more fungi selected from the genera Candida or Cryptococcus, or molds.

[0121] Other specific bacteria that may be assayed using the methods and systems disclosed herein include Staphylococcus aureus, Staphylococcus lugdunensis, coagulase-negative Staphylococcus species (including, but not limited to, undifferentiated Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus capitis), Enterococcus faecalis, Enterococcus faecium (undifferentiated Enterococcus faecium, and other Enterococcus species). spp., but excluding Enterococcus faecalis), Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus species (undifferentiated Streptococcus mitis, Streptococcus pyogenes, Streptococcus gallolyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus gallolyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus gallolyticus, Streptococcus pneumoniae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus pneumoniae ... pneumoniae), Pseudomonas aeruginosa, Acinetobacter baumannii, Klebsiella spp.), including but not limited to undifferentiated Klebsiella pneumoniae and Klebsiella oxytoca, Escherichia coli, Enterobacter spp., including but not limited to undifferentiated Enterobacter cloacae and Enterobacter aerogenes, Proteus spp., including but not limited to undifferentiated Proteus mirabilis and Proteus vulgaris, Citrobacter spp., including but not limited to undifferentiated Citrobacter freundii, freundii, Citrobacter koseri, Serratia marcescens, Candida albicans, Candida glabrata, and Candida tropicalis.

[0122] Other more specific bacteria that can be assayed include Acinetobacter baumannii, Actinobacillus spp., Actinomyces spp. (including but not limited to Actinomyces israelii and Actinomyces naeslundii), Aeromonas spp. (including but not limited to Aeromonas hydrophila, Aeromonas veronii biovar sobria (Aeromonas sobria), and Aeromonas caviae), Anaplasma phagocytophilum, and the like. phagocytophilum, Alcaligenes xylosoxidans, Actinobacillus actinomycetemcomitans, Bacillus (including, but not limited to, Bacillus anthracis, Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, and Bacillus stearothermophilus), Bacterioides (including, but not limited to, Bacteroides fragilis), Bartonella (including, but not limited to, Bartonella baciliformis), bacilliformis and Bartonella henselae), Bifidobacterium species, Bordetella species (Bordetella pertussis, Bordetella parapertussis,parapertussis, and Bordetella bromchiseptica, Borrelia spp. (including but not limited to Borrelia recurrentis and Borrelia burgdorferi), Brucella spp. (including but not limited to Brucella abortus, Brucella canis, Brucella melintensis, and Brucella suis), Burkholderia spp. (including but not limited to Burkholderia pseudomallei and Burkholderia cepacia), Campylobacter spp. (including but not limited to Campylobacter jejuni, Campylobacter coli, Campylobacter cholera, and Campylobacter cholera), coli, Campylobacter lari and Campylobacter fetus, Capnocytophaga species, Cardiobacterium hominis, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Citobacter species, Coxiella burnetti, Corynebacterium species (Corynebacterium diphthariae, Corynebacterium jacqueum), jeikeum and Corynebacterium), Clostridium species (Clostridium perfringens, Clostridium difficile,difficile, Clostridium botulinum and Clostridium tetani (Clostridium tetani), Eikenella corrodens, Enterobacter species (Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae and Escherichia coli (including but not limited to opportunistic E. coli, enterotoxigenic E. coli, enteroinvasive E. coli, enteropathogenic E. coli, enterohemorrhagic E. coli, enteroaggregative E. coli and uropathogenic E. coli), Enterococcus species (Enterococcus faecalis, Enter ... faecalis and Enterococcus faecium, Ehrlichia species (including but not limited to Ehrlichia chafeensia and Ehrlichia canis), Erysipelothryx rhusiopathiae (Erysipelothrix rhusiopathiae), Eubacterium species, Francisella tularensis (Fusobacterium nucleatum), Gardnerella vaginalis, Gemella morbillorum, Haemophilus species (Haemophilus influenzae), influenzae, Haemophilus ducreyi, Haemophilus parainfluenzae, Haemophilus haemolyticusHelicobacter spp. (Helicobacter pylori, Helicobacter cinaedi, and Helicobacter fennelliae, Kingella kingii, Klebsiella spp. (Klebsiella pneumoniae, Klebsiella granulomatis, and Klebsiella oxytoca), Lactobacillus spp., Listeria monocytogenes, Leptospira interrogans, Legionella pneumophila, pneumophila, Leptospira interrogans, Peptostreptococcus spp., Moraxella catarrhalis, Morganella spp., Mobiluncus spp., Micrococcus spp., Mycobacterium spp. (including, but not limited to, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium bovis, and Mycobacterium marinum), Mycoplasma spp. (Mycoplasma pneumoniae, Mycoplasma hominis, Mycoplasma hominis and Mycoplasma genitalium, Nocardia species (Nocardia asteroides,asteroides, Nocardia cyriacigeorgica and Nocardia brasiliensis, Neisseria spp. (including but not limited to Neisseria gonorrhoeae and Neisseria meningitidis), Pasteurella multocida, Plesiomonas shigelloides, Prevotella spp., Porphyromonas spp., Prevotella melaninogenica, Proteus spp. (Proteus vulgaris and Proteus mirabilis), mirabilis), Providencia species (including but not limited to Providencia alcalifaciens, Providencia rettgeri, Providencia stuartii), Pseudomonas aeruginosa, Propionibacterium acnes, Rhodococcus equi, Rickettsia species (including but not limited to Rickettsia rickettsii, Rickettsia akari, and Rickettsia prowazekii), Orientia tsutsugamushi. (formerly known as Rickettsia tsutsugamushi), and Rickettsia typhityphi), Rhodococcus, Stenotrophomonas maltophilia, Salmonella spp. (including but not limited to Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, Salmonella cholerasuis, and Salmonella typhimurium), Serratia spp. (including but not limited to Serratia marcesans and Serratia liquifaciens), Shigella spp. (Shigella dysenteriae), dysenteriae, Shigella flexneri, Shigella boydii and Shigella sonnei, Staphylococcus species (including but not limited to Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus hemolyticus, Staphylococcus saprophyticus), Streptococcus species (including but not limited to Streptococcus pneumoniae,pneumoniae) (e.g., chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, spectinomycin-resistant serotype 6B Streptococcus pneumoniae, streptomycin-resistant serotype 9V Streptococcus pneumoniae, erythromycin-resistant serotype 14 Streptococcus pneumoniae, opisthotin-resistant serotype 14 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, tetracycline-resistant serotype 19F Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, and trimethoprim-resistant serotype 23F Streptococcus pneumoniae), chloramphenicol-resistant serotype 4 Streptococcus pneumoniae, rifampicin-resistant serotype 18C Streptococcus pneumoniae, penicillin-resistant serotype 19F Streptococcus pneumoniae, or trimethoprim-resistant serotype 23F Streptococcus pneumoniae), Streptococcus agalactiae (Streptococcus agalactiae, Streptococcus mutans, Streptococcus pyogenes, Group A Streptococci, Streptococcus pyogenesis (Streptococcus pyogenes), Group B Streptococci, Streptococcus agalactiae, Group C Streptococci, Streptococcus antinosus, Streptococcus equismilis, Group G Streptococci, Streptococcus bovis, Group F Streptococci Streptococci, Streptococcus anginosus and Group G Streptococci, Spirillum minus, Streptobacillus moniliformi, Treponema species (Treponema Treponema carateum, Treponema petenue, Treponema pallidum and Treponema endemicum, Tropheryma whippelii, Ureaplasma urealyticum, Veillonella spp, Vibrio spp (Vibrio cholerae, Vibrio parahemolyticus, Vibrio vulnificus, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginolyticus, Vibrio mimicus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela and Vibrio furnisii, Yersinia species (including but not limited to Yersinia enterocolitica, Yersinia pestis and Yersinia pseudotuberculosis and Xanthomonas maltophilia).

[0123] Additionally, other microorganisms or infectious agents that can be assayed using the methods and systems disclosed herein include Candida species (including, but not limited to, Candida albicans, Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei), Aspergillus species (including, but not limited to, Aspergillus fumigatous, Aspergillus flavus, and Aspergillus clavatus), Cryptococcus species (including, but not limited to, Cryptococcus neoformans, Cryptococcus gattii, and Cryptococcus spp.), and / or other pathogens that can be assayed using the methods and systems disclosed herein. gattii, Cryptococcus laurentii and Cryptococcus albidus), Fusarium species (including but not limited to Fusarium oxysporum, Fusarium solani, Fusarium verticillioides and Fusarium proliferatum), Rhizopus oryzae, Penicillium marneffei, Coccidiodes immitis and Blastomyces dermatitidis.

[0124] The sample vessel 104 may be made in part of an inert or non-conductive material. In some embodiments, the sample vessel 104 may be made in part of a polymeric material, a ceramic material, or glass, or a combination thereof. As more specific examples, the sample vessel 104 may include or be made in part of polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), or a combination thereof.

[0125] The sensor apparatus 100 may also include a reference sensor member 108 and an active sensor member 122. The reference sensor member 108 may further include a reference conduit 110 that includes a reference conduit cavity 112, a reference conduit first opening 114, and a reference conduit second opening 116.

[0126] The reference conduit 110 may have a conduit wall 138 and may gradually taper from the reference conduit proximal end 140 to the reference conduit distal end 142. In one embodiment, the reference conduit 110 may be substantially shaped as a cone or a cone shape with the reference conduit cavity 112 also substantially shaped as a cone or a cone shape. In another embodiment, the reference conduit 110 may be substantially shaped as an elongated pyramid with a polygonal base. For example, the reference conduit 110 may be substantially shaped as an elongated triangular pyramid, a square pyramid, or a pentagonal pyramid. In additional embodiments, the reference conduit 110 may be substantially shaped as a cylinder with a cylindrical reference conduit cavity 112. In these embodiments, the reference conduit 110 may also have a tapered reference conduit distal end 142.

[0127] The reference conduit cavity 112 can be at least partially filled with a reference buffer gel 118. The reference conduit 110 can extend into the sample chamber 106 such that a second opening 116 of the reference conduit is configured to allow the sample 102 to be in fluid contact with the reference buffer gel 118 in the reference conduit cavity 112. For example, the reference conduit second opening 116 can be in liquid-solid contact with the reference buffer gel 118. A salt bridge can thus be formed with a liquid-solid interface acting as the junction (interface) of the salt bridge.

[0128] In one embodiment, the reference buffer gel 118 can be an agar gel formed from agar powder mixed with a reference buffer. In another embodiment, the reference buffer gel 118 can be a gel formed from another type of polysaccharide mixed with a reference buffer gel solution. As a more specific example, the agar powder used was an agar powder provided by Sigma-Aldrich (e.g., Sigma-Aldrich TM Agar Powder 05040) or agar powder provided by Thermo Fisher Scientific (e.g., Fisher Scientific TM The agar powder may be commercially available agar powder such as Agar Powder, catalog number S14153).

[0129] The reference buffer may be an aqueous redox buffer. In one embodiment, the reference buffer may be a redox aqueous buffer including deionized water, potassium ferricyanide, dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof. For example, the reference buffer may be an aqueous redox buffer including deionized water (e.g., about 95% to 99%), potassium ferricyanide (III) (e.g., about 0.1% to 0.9%), potassium ferricyanide (II) (e.g., about 0.1% to 99%), potassium dihydrogen phosphate (e.g., less than about 0.5%), and disodium hydrogen phosphate (less than about 0.5%). As a more specific example, the reference buffer may be 220 mV / pH 7 redox buffer (product code 51350060) provided by Mettler-Toledo AG.

[0130] In other embodiments, the reference buffer can be an aqueous redox buffer containing 3 M KCl. As a more specific example, the reference buffer can be 3 M KCl Redox Buffer provided by Mettler-Toledo AG (Part No. 63056165).

[0131] In one embodiment, the reference buffer gel 118 may include agar powder at a concentration of about 1% (w / v%, g / mL). In another embodiment, the reference buffer gel 118 may include agar powder at a concentration of about 5% (w / v%, g / mL). In some embodiments, the reference buffer gel 118 may include agar powder at a concentration of about 1% (w / v%, g / mL) to 5% (w / v%, g / mL). In other embodiments, the reference buffer gel 118 may include agar powder at a concentration of about 5% (w / v%, g / mL) to 10% (w / v%, g / mL).

[0132] The reference buffer gel 118 can be made by heating an aqueous reference buffer above its boiling point and stirring agar powder into the heated aqueous reference buffer. Once the agar powder is completely dissolved in the heated aqueous buffer, the hot gel slurry can be poured into the reference electrode cavity 112 and allowed to cool to room temperature. The reference buffer gel 118 can solidify as the hot gel cools to room temperature in the reference conduit cavity 112.

[0133] In one embodiment, the reference conduit cavity 112 can be filled with about 100 μL of reference buffer gel 118. In another embodiment, the reference conduit cavity 112 can be filled with about 500 μL of reference buffer gel 118. In another embodiment, the reference conduit cavity 112 can be filled with about 100 μL to about 500 μL of reference buffer gel 118. In a further embodiment, the reference conduit cavity 112 can be filled with about 500 μL to about 1 mL of reference buffer gel 118. The amount of reference buffer gel 118 can depend on the volume of the reference conduit cavity 112, the size of the reference conduit 110, the size of the sample vessel 104, or a combination thereof.

[0134] The reference sensor member 108 may also include a reference electrode 120 that extends into the reference conduit cavity 112 that is filled with a reference buffer gel 118. The reference electrode 120 may extend partially into the reference conduit cavity 112 that is filled with the reference buffer gel 118.

[0135] As a more specific example, the reference electrode 120 can include a reference electrode tip 144. The reference electrode tip 144 can be positioned proximate to the reference conduit second opening 116 when the reference electrode 120 extends partially into the reference conduit cavity 112 filled with the reference buffer gel 118. More specifically, a volume of the reference buffer gel 118 can separate the reference electrode tip 144 from the reference conduit second opening 116. The location of the reference electrode 120 within the reference conduit cavity 112 will be described in more detail in the next section.

[0136] The reference electrode 120 can be made in part of one or more metals. In one embodiment, the reference electrode 120 can be made of platinum (Pt), stainless steel, or a combination thereof. In other embodiments, the reference electrode 120 can be made in part of another type of conductive metal. For example, the reference electrode 120 can be a metallic pin, wire, or rod inserted into the reference buffer gel 118 such that a segment of the reference electrode 120 (e.g., the reference electrode tip 144 and a segment of the reference electrode 120 proximate to the reference electrode tip 144) penetrates and resides within the reference buffer gel 118.

[0137] One unexpected discovery made by the present application is that the reference buffer gel 118 slows diffusion across the liquid-solid interface created by the reference buffer gel 118 and the sample 102, and that such a reference sensor element 108 can be used as an effective reference cell for pH measurements.

[0138] The sensor device 100 may also include an active sensor member 122 that includes an active electrode housing 124 that extends into the sample chamber 106 configured to receive the sample 102. The active sensor member 122 may also include an active electrode 126 that is at least partially contained or surrounded by the active electrode housing 124.

[0139] The active electrode housing 124 can be made of an inert or non-conductive material and can include polymeric materials such as polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), ceramic, silicon dioxide (SiO2), or various types of glass, or combinations thereof.

[0140] The active electrode 126 can include a sample contacting surface 128. The active electrode 126 can be disposed at a distal end 130 of the active member. The active electrode housing 124 can be configured to expose the sample contacting surface 128 such that the sample contacting surface 128 is in fluid communication with the sample 102.

[0141] In some embodiments, the active electrode 126 can be made in part of a metal oxide. For example, the active electrode 126 can be made in part of silicon dioxide (SiO2), aluminum oxide (Al2O3), titanium dioxide (TiO2), tantalum pentoxide (Ta2O5), hafnium dioxide (HfO2), iridium dioxide (IrO2), ruthenium dioxide (RuO2), zirconium dioxide (ZrO2), or combinations thereof.

[0142] The active sensor member 122 may include a conductive contact layer 132 coupled to and positioned proximate to the active electrode 126. The conductive contact layer 132 may be contained within or encased by the active electrode 124.

[0143] The conductive contact layer 132 may be made in part of one or more metals. In some embodiments, the conductive contact layer 132 may be made of aluminum (Al), copper (Cu), platinum (Pt), or a combination thereof. The conductive contact layer 132 and the reference electrode 120 may be electrically coupled to the voltmeter 136 by a conductive connection 134 or conductive wire. The conductive connection 134 may be made in part of a conductive material. In one embodiment, the conductive connection 134 may be a copper wire. For example, the conductive connection 134 may be electrodeposited copper, rolled annealed copper, highly ductile electrodeposited copper, or a combination thereof. In other embodiments, the conductive connection 134 may be made in part of silver or nickel.

[0144] In some embodiments, the voltmeter 136 can be a high impedance voltmeter. The conductive connection 134 can electrically couple the active electrode 126 (via the conductive contact layer 132) and the reference electrode 120 to the voltmeter 136. The pH of the sample 102 can be measured based on the potential difference between the active electrode 126 and the reference electrode 120.

[0145] The sensor device 100 may also include an aeration conduit 146. A segment of the aeration conduit 146 may extend into the sample chamber 106 such that a lumen or cavity within the aeration conduit 146 is in fluid communication with the sample chamber 106. The aeration conduit 146 may be configured to aerate the sample 102 contained within the sample container 104. Aeration of the sample 102 may increase the growth rate of microorganisms within the sample 102 by increasing the supply of oxygen to the microorganisms. Additionally, aeration may allow detachment of microorganisms from the inner walls of the sample container 104 to inhibit biofilm formation.

[0146] Figure 1B illustrates one embodiment of a sensor device 101 for measuring the oxidation-reduction potential (ORP) of a sample 102. Figure 1B illustrates a side cross-sectional view of the sensor device 101.

[0147] The sensor device 101 can include a sample container 104 having a sample chamber 106 configured to receive a sample 102. The sample 102 can be the same sample 102 described with respect to the sensor device 100 (see FIG. 1A). The sample 102 can include a number of microorganisms or infectious agents. The devices, systems and methods disclosed herein can be used to analyze the sample 102 for microbial growth or lack thereof as part of a microbiological assay or antibiotic susceptibility testing (AST) method.

[0148] In some embodiments, the aqueous growth medium can be added to the sample 102 prior to introduction into the sample vessel 104. In other embodiments, the aqueous growth medium can be added to the sample 102 after the sample 102 has been injected, delivered or otherwise introduced into the sample vessel 104.

[0149] In one embodiment, the aqueous growth medium can be glucose-supplemented Mueller Hinton Broth (MHG), in other embodiments, the aqueous growth medium can be a solution containing Bacto-tryptone, tryptic soy digest, yeast extract, beef extract, cation-adjusted Muller Hinton Broth (CAMHB), starch, acid hydrolysate of casein, calcium chloride, magnesium chloride, sodium chloride, blood or hemolysate such as horse hemolysate (LHB), CAMHB-LHB, glucose or other carbohydrates, or combinations thereof.

[0150] The sample vessel 104 may be made in part of an inert or non-conductive material. In some embodiments, the sample vessel 104 may include or be made in part of a polymeric material, a ceramic material, or glass, or a combination thereof. As more specific examples, the sample vessel 104 may comprise or be made in part of polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), or a combination thereof.

[0151] The sensor device 101 may also include a reference sensor member 108 and an active sensor member 122. The reference sensor member 108 may further include a reference conduit 110 that includes a reference conduit cavity 112, a reference conduit first opening 114, and a reference conduit second opening 116.

[0152] The reference conduit 110 can have a conduit wall 138. The conduit wall 138 can be gradually tapered from the reference conduit proximal end 140 to the reference conduit distal end 142. In one embodiment, the reference conduit 110 can be substantially shaped as a cone or frustoconic with the reference conduit cavity 112 substantially shaped as a cone or frustoconic. In another embodiment, the reference conduit 110 can be substantially shaped as an elongated pyramid with a polygonal base. For example, the reference conduit 110 can be substantially shaped as an elongated triangular pyramid, a square pyramid, or a pentagonal pyramid. In additional embodiments, the reference conduit 110 can be substantially shaped as a cylinder with a substantially cylindrical reference conduit cavity 112. In these embodiments, the reference conduit 110 can also have a tapered reference conduit distal end 142.

[0153] The reference conduit cavity 112 can be at least partially filled with a reference buffer gel 118. The reference conduit 110 can extend into the sample chamber 106 such that a second opening 116 of the reference conduit is configured to allow the sample 102 to be in fluid contact with the reference buffer gel 118 in the reference conduit cavity 112. For example, the second opening 116 of the reference conduit can allow the sample 102 to be in liquid-solid contact with the reference buffer gel 118.

[0154] In one embodiment, the reference buffer gel 118 can be an agar gel formed from agar powder mixed with a reference buffer. In another embodiment, the reference buffer gel 118 can be an agar gel formed from agar powder mixed with a reference buffer. In another embodiment, the reference buffer gel 118 can be a gel formed from another type of polysaccharide mixed with a reference buffer. As a more specific example, the agar powder used can be an agar powder provided by Sigma-Aldrich (e.g., Sigma-Aldrich TM Agar Powder 05040) or agar powder provided by Thermo Fisher Scientific (e.g., Fisher Scientific TM The agar powder can be commercially available agar powder such as Agar Powder (catalog number S14153).

[0155] The reference buffer may be an aqueous redox buffer. In one embodiment, the reference buffer may be a redox aqueous buffer comprising deionized water, potassium ferricyanide, dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof. For example, the reference buffer may be an aqueous redox buffer comprising deionized water (e.g., about 95% to 99%), potassium ferricyanide (III) (e.g., about 0.1% to 0.9%), potassium ferricyanide (II) (e.g., about 0.1% to 0.9%), potassium dihydrogen phosphate (e.g., less than about 0.5%), and disodium hydrogen phosphate (less than about 0.5%). As a more specific example, the reference buffer may be 220 mV / pH 7 redox buffer provided by Mettler-Toledo (product number 51350060).

[0156] In another embodiment, the reference buffer can be an aqueous redox buffer containing 3 M KCl. As a more specific example, the reference buffer can be 3 M KCl redox buffer provided by Mettler-Toledo (product number 63056165).

[0157] In one embodiment, the reference buffer gel 118 can include agar powder at a concentration of about 1% (w / v%, g / mL). In another embodiment, the reference buffer gel 118 can include agar powder at a concentration of about 5% (w / v%, g / mL). In some embodiments, the reference buffer gel 118 can include agar powder at a concentration of about 1% (w / v%, g / mL) to 5% (w / v%, g / mL). In other embodiments, the reference buffer gel 118 can include agar powder at a concentration between about 5% (w / v%, g / mL) and 10% (w / v%, g / mL).

[0158] The reference buffer gel 118 can be made by heating an aqueous buffer solution above the boiling point of the aqueous buffer solution and heating agar powder in the heated aqueous buffer solution. After the agar powder is completely dissolved in the heated aqueous buffer solution, the hot gel slurry can be poured into the reference conduit cavity 112 and allowed to cool to room temperature. As the hot gel cools to room temperature in the reference conduit cavity 112, the reference buffer gel 118 can solidify.

[0159] In one embodiment, the reference conduit cavity 112 can be filled with about 100 μL of reference buffer gel 118. In another embodiment, the reference conduit cavity 112 can be filled with about 500 μL of reference buffer gel 118. In another embodiment, the reference conduit cavity 112 can be filled with about 100 μL to about 500 μL of reference buffer gel 118. In a further embodiment, the reference conduit cavity 112 can be filled with about 500 μL to about 1 mL of reference buffer gel 118. The amount of reference buffer gel 118 can depend on the volume of the reference conduit cavity 112, the size of the reference conduit 110, the size of the sample vessel 104, or a combination thereof.

[0160] The reference sensor member 108 may also include a reference electrode 120 that extends into the reference conduit cavity 112 that is filled with a reference buffer gel 118. The reference electrode 120 may extend partially into the reference conduit cavity 112 that is filled with the reference buffer gel 118.

[0161] As a more specific example, the reference electrode 120 may include a reference electrode tip 144. The reference electrode tip 144 may be positioned proximal to the reference conduit second opening 116 when the reference electrode 120 extends partially into the reference conduit cavity 112 filled with the reference buffer gel 118. More specifically, a volume of the reference buffer gel 118 may separate the reference electrode tip 144 from the reference conduit second opening 116. The location of the reference electrode 120 within the reference conduit cavity 112 will be discussed in more detail in the next section.

[0162] The reference electrode 120 can be made in part of one or more metals. In one embodiment, the reference electrode 120 can be made in part of platinum (Pt), stainless steel, or a combination thereof. In another embodiment, the reference electrode 120 can be made in part of another type of conductive material. For example, the reference electrode 120 can be a metallic pin, wire, or rod inserted into the reference buffer gel 118 such that a segment of the reference electrode 120 (e.g., the reference electrode tip 144 and a segment of the reference electrode 120 proximal to the reference electrode tip 144) penetrates and resides within the reference buffer gel 118.

[0163] One unexpected discovery made by the present application is that the reference buffer gel 118 slows diffusion across the liquid-solid interface formed by the reference buffer gel 118 and the sample 102, and extends the amount of time that such a reference sensor element 108 can be used as an effective reference cell for ORP measurements.

[0164] The sensor device 101 may also include an active sensor member 122 that includes an active electrode housing 124 that extends into a sample chamber 106 configured to receive a sample 102. The active sensor member 122 may also include an active electrode 148 that is at least partially contained or surrounded by the active electrode housing 124.

[0165] The active electrode housing 124 can be made of an inert or non-conductive material and can include polymeric materials such as polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), ceramic, silicon dioxide (SiO2), or various types of glass, or combinations thereof.

[0166] The active electrode 148 may include a sample contacting surface 128. The active electrode 148 may be disposed at the active member distal end 130. The active electrode housing 124 may be configured to expose the sample contacting surface 128 such that the sample contacting surface 128 is in fluid communication with the sample 102.

[0167] In some embodiments, the active electrode 148 can be made in part of one or more metals, one or more metal oxides, or a combination thereof. For example, the active electrode 148 can be made in part of platinum (Pt), gold (Au), a redox-sensitive metal oxide, or a combination thereof. In some embodiments, the redox-sensitive metal oxide can include silicon dioxide (SiO2), aluminum oxide (Al2O3), titanium dioxide (TiO2), tantalum pentoxide (Ta2O5), hafnium dioxide (HfO2), iridium dioxide (IrO2), ruthenium dioxide (RuO2), zirconium dioxide (ZrO2), or a combination thereof.

[0168] The active electrode 148 and the reference electrode 120 may be electrically coupled to the voltmeter 136 by a conductive connection 134 or conductive wire. The conductive connection 134 may be made in part of a conductive material. In one embodiment, the conductive connection 134 may be a copper wire. For example, the conductive connection 134 may be electrodeposited copper, rolled annealed copper, highly ductile electrodeposited copper, or a combination thereof. In other embodiments, the conductive connection 134 may be made in part of silver or nickel.

[0169] In some embodiments, the voltmeter 136 may be a high impedance voltmeter. The ORP of the sample 102 may be measured based on the potential difference between the active electrode 148 and the reference electrode 120.

[0170] The sensor device 101 may also include an aeration conduit 146. A segment of the aeration conduit 146 may extend into the sample chamber 106 such that a lumen or cavity within the aeration conduit 146 is in fluid communication with the sample chamber 106. The aeration conduit 146 may be configured to aerate the sample 102 received within the sample container 104. Aeration of the sample 102 may increase the growth rate of microorganisms within the sample 102 by increasing the supply of oxygen to the microorganisms. Additionally, aeration may allow detachment of microorganisms from the inner walls of the sample container 104 to inhibit biofilm formation.

[0171] Figure 1C illustrates another embodiment of a sensor apparatus 103 for measuring the pH of a sample 102. Figure 1C illustrates a side cross-sectional view of the sensor apparatus 103.

[0172] The sensor device 103 can include a sample container 104 having a sample chamber 106 configured to receive a sample 102. The sample 102 can be the same sample 102 described with respect to the sensor device 100 (as shown in FIG. 1A) and the sensor device 101 (as shown in FIG. 1B). The sample 102 can include a number of microorganisms or infectious agents. The devices, systems, and methods disclosed herein can be used to analyze the sample 120 for microbial growth or lack thereof as part of a microbiological assay or antibiotic susceptibility testing (AST) method.

[0173] In some embodiments, the aqueous growth medium can be added to the sample 102 prior to introduction into the sample vessel 104. In other embodiments, the aqueous growth medium can be added to the sample 102 after the sample 102 has been injected, delivered or otherwise introduced into the sample chamber 104.

[0174] In one embodiment, the aqueous growth medium can be Muller Hinton Broth (MHG) supplemented with glucose, in another embodiment, the aqueous growth medium can be a solution containing bactotryptone, tryptic soy digest, yeast extract, beef extract, cation adjusted Muller Hinton Broth (CAMHB), starch, acid hydrolysate of casein, calcium chloride, magnesium chloride, sodium chloride, blood or hemolyzed blood such as horse hemolyzed blood (LHB), CAMHB-LHB, glucose or other carbohydrates, or combinations thereof.

[0175] The sample vessel 104 may be made in part of an inert or non-conductive material. In some embodiments, the sample vessel 104 may be made of a polymeric material, a ceramic material, or glass, or a combination thereof. As more specific examples, the sample vessel 104 may include or be made in part of polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), or a combination thereof.

[0176] The sensor device 103 may also include a reference sensor member 108 and an active sensor component 122. The reference sensor member 108 may further include a reference conduit 110 including a reference conduit proximal end 140 defining a reference conduit first opening 114, a reference conduit distal end 142 defining a reference conduit second opening 116, and a reference conduit cavity 112 between the reference conduit proximal end 140 and the reference conduit distal end 142.

[0177] The reference conduit 110 can include a conduit wall 138, and the conduit wall 138 can be gradually tapered from the reference conduit proximal end 140 to the reference conduit distal end 142. In one embodiment, the reference conduit 110 can be shaped as a cone or frustoconic, with the reference conduit cavity 112 shaped as a substantially cone or frustoconic. In another embodiment, the reference conduit 110 can be shaped substantially as an elongated pyramid with a polygonal base, for example, the reference conduit 110 can be shaped substantially as an elongated triangular pyramid, square pyramid, or pentagonal pyramid. In additional embodiments, the reference conduit 110 can be shaped substantially as a cylinder with a substantially cylindrical reference conduit cavity 112. In these embodiments, the reference conduit 110 can have a tapered reference conduit distal end 142.

[0178] The reference conduit 110 can extend into the sample chamber 106 such that at least a portion of the reference conduit 110 is submerged in the sample 102 when the sample chamber 106 is filled with the sample 102. For example, the reference conduit distal end 142 can be submerged in the sample 102 in the sample chamber 106.

[0179] The reference sensor member 108 may include an ion exchange membrane 150 coupled to the reference conduit distal end 142. The ion exchange membrane 150 may be configured to block the reference conduit second opening 116 such that microorganisms within the sample 102 do not enter the reference conduit cavity 112. However, the ion exchange membrane 150 allows ions to penetrate the ion exchange membrane 150 to maintain charge balance.

[0180] In some embodiments, the ion exchange membrane 150 can be a perfluorosulfonated ionomer membrane. As a more specific example, the ion exchange membrane 150 can be a Nafion® membrane provided by EI du Pont de Nemours and Company. Also, for example, the ion exchange membrane 150 can be an Aciplex® membrane provided by Asahi Kasei Corporation or a Flemion® membrane provided by AGC Chemical Americas, Inc.

[0181] In some embodiments, the ion exchange membrane 150 can have a thickness of about 25 μm to about 180 μm. More specifically, the ion exchange membrane 150 can have a thickness of about 25 μm to about 100 μm. The ion exchange membrane 150 can also have a thickness of about 100 μm to about 180 μm.

[0182] In another embodiment, the ion exchange membrane 150 can have a thickness of about 180 μm to about 200 μm. For example, the ion exchange membrane can have a thickness of about 183 μm.

[0183] The reference conduit cavity 112 can be at least partially filled with a reference buffer 152. The reference buffer 152 can be an aqueous redox buffer.

[0184] In one embodiment, the reference buffer 152 can include deionized water, potassium ferricyanide, dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof. For example, the reference buffer 152 can include about 95%-99% (v / v%) deionized water, about 0.1%-0.9% (w / v%) potassium ferricyanide, about 0.1%-0.9% (w / v%) potassium ferricyanide, less than about 0.5% (w / v%) potassium dihydrogen phosphate, and less than about 0.5% (w / v) disodium hydrogen phosphate. As a more specific example, the reference buffer can be 3 M KCl Redox Buffer (Part No. 63056165) provided by Mettler-Toledo.

[0185] In another embodiment, the reference buffer 152 can be an aqueous redox buffer containing 3 M KCl. As a more specific example, the reference buffer can be a 3 M KCl redox buffer provided by Mettler-Toledo.

[0186] In one embodiment, the reference conduit cavity 112 can be filled with about 100 μL of the reference buffer 152. In another embodiment, the reference conduit cavity 112 can be filled with about 500 μL of the reference buffer 152. In another embodiment, the reference conduit cavity 112 can be filled with about 100 μL to about 500 μL of the reference buffer 152. In a further embodiment, the reference conduit cavity 112 can be filled with about 500 μL to about 1 mL of the reference buffer 152. The amount of the reference buffer 152 can depend on the volume of the reference conduit cavity 112, the size of the reference conduit 110, the size of the sample vessel 104, or a combination thereof.

[0187] The reference sensor member 108 may also include a reference electrode 120 extending into a reference conduit cavity 112 filled with a reference buffer solution 152. The reference electrode 120 may extend partially into the reference conduit cavity 112.

[0188] As a more specific example, the reference electrode 120 can include a reference electrode tip 144. The reference electrode tip 144 can be positioned proximal to the ion exchange membrane 150 when the reference electrode 120 extends partially into the reference conduit cavity 112 filled with a reference buffer 152. More specifically, a volume of the reference buffer 152 can separate the reference electrode tip 144 from the ion exchange membrane 150. The location of the reference electrode 120 within the reference conduit cavity 112 will be discussed in the next section.

[0189] The reference electrode 120 can be made in part of one or more metals. In one embodiment, the reference electrode 120 can be made in part of platinum (Pt), stainless steel, or a combination thereof. In other embodiments, the reference electrode 120 can be made in part of another type of conductive metal. For example, the reference electrode 120 can be a metallic pin, wire, or rod such that a segment of the reference electrode 120 (e.g., the reference electrode tip 144 and a segment of the reference electrode 120 proximal to the reference electrode tip 144) is immersed in the reference buffer 152.

[0190] The sample 102 can be in fluid contact with the ion exchange membrane 150 such that the ion exchange membrane acts as a salt bridge interface. Ions can diffuse from one side of the ion exchange membrane 150 to the other to maintain charge balance.

[0191] The sensor device 103 may also include an active sensor member 122 that includes an active electrode housing 124 that extends into a sample chamber 106 configured to receive a sample 102. The active sensor member 122 may also include an active electrode 126 that is at least partially contained or surrounded by the active electrode housing 124.

[0192] The active electrode 124 can be made of an inert or non-conductive material. The active electrode housing 124 can include polymeric materials such as polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), ceramics, silicon dioxide (SiO2), or various types of glass, or combinations thereof.

[0193] The active electrode 126 can include a sample contacting surface 128. The active electrode 126 can be disposed at a distal end 130 of the active member. The active electrode housing 124 can be configured to expose the sample contacting surface 128 such that the sample contacting surface 128 is in fluid communication with the sample 102.

[0194] In some embodiments, the active electrode 126 can be made in part of a metal oxide. For example, the active electrode 126 can be made in part of silicon dioxide (SiO2), aluminum oxide (Al2O3), titanium dioxide (TiO2), tantalum pentoxide (Ta2O5), hafnium dioxide (HfO2), iridium dioxide (IrO2), ruthenium dioxide (RuO2), zirconium dioxide (ZrO2), or combinations thereof.

[0195] The active sensor member 122 may also include a conductive contact layer 132 coupled to and positioned proximate to the active electrode 126. The conductive contact layer 132 may be contained within or surrounded by the active electrode housing 124.

[0196] The conductive contact layer 132 may be made in part of one or more metals. In some embodiments, the conductive contact layer 132 may be made in part of aluminum (Al), copper (Cu), platinum (Pt), or a combination thereof. The conductive contact layer 132 and the reference electrode 120 may be electrically coupled to the voltmeter 136 by a conductive connection 134 or conductive wire. The conductive connection 134 may be made in part of a conductive material. In one embodiment, the conductive connection 134 may be a copper wire. For example, the conductive connection 134 may be electroplated copper, rolled annealed copper, highly ductile electroplated copper, or a combination thereof. In other embodiments, the conductive connection 134 may be made in part of silver or nickel.

[0197] In some embodiments, the voltmeter 136 can be a high impedance voltmeter. The conductive connection 134 can electrically couple the active electrode 126 (via the conductive contact layer 132) and the reference electrode 120 to the voltmeter 136. The pH of the sample 102 can be measured based on the potential difference between the active electrode 126 and the reference electrode 120.

[0198] The sensor device 103 can include an aeration conduit 146. A segment of the aeration conduit 146 can extend into the sample chamber 106 such that a lumen or cavity in the aeration conduit 146 is in fluid communication with the sample chamber 106. The aeration conduit 146 can be configured to aerate the sample 102 received in the sample container 104. Aeration of the sample 102 can increase the growth rate of microorganisms in the sample 102 by increasing the supply of oxygen to the microorganisms. Additionally, aeration can also allow detachment of microorganisms from the inner walls of the sample container 104 to inhibit biofilm formation.

[0199] Figure ID illustrates another embodiment of a sensor device 105 for measuring the ORP of a sample 102. Figure ID illustrates a side cross-sectional view of the sensor device 105.

[0200] The sensor device 105 can include a sample container 104 having a sample chamber 106 configured to receive a sample 102. The sample can be the same sample 102 discussed with respect to sensor device 100 (shown in FIG. 1A), sensor device 101 (shown in FIG. 1B), and sensor device 103 (shown in FIG. 1C). The sample 102 can include a number of microorganisms or infectious agents. The devices, systems, and methods disclosed herein can be used to analyze microbial growth or lack thereof as part of a microbiological assay or antibiotic susceptibility testing (AST) method.

[0201] In some embodiments, the aqueous growth medium can be added to the sample 102 prior to introduction into the sample vessel 104. In other embodiments, the aqueous growth medium can be added to the sample 102 after the sample 102 has been injected, delivered, or otherwise introduced into the sample vessel 104.

[0202] In some embodiments, the aqueous growth medium can be Mueller Hinton Broth (MHG) supplemented with glucose, in other embodiments, the aqueous growth medium can be a solution containing bactotryptone, tryptic soy digest, yeast extract, beef extract, cation adjusted Mueller Hinton Broth (CAMHB), starch, acid hydrolysate of casein, calcium chloride, magnesium chloride, sodium chloride, blood or hemolysate including horse hemolysate (LHB), CAMHB-LHB, glucose or other carbohydrates, or combinations thereof.

[0203] The sensor device 105 can include a sample container 104 having a sample chamber 106 configured to receive a sample 102. The sample 102 can be the same sample 102 discussed with respect to the sensor device 100 (shown in FIG. 1A) and the sensor device 101 (shown in FIG. 1B). The sample 102 can include a number of microorganisms or infectious agents. The devices, systems, and methods disclosed herein can be used to analyze microbial growth or lack thereof as part of a microbiological assay or antibiotic susceptibility testing (AST) method.

[0204] The sample vessel 104 may be made in part of an inert or non-conductive material. In some embodiments, the sample vessel 104 may include or be made in part of a polymeric material, a ceramic material, or glass, or a combination thereof. As more specific examples, the sample vessel 104 may include or be made in part of polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), or a combination thereof.

[0205] The sensor device 105 may also comprise a reference sensor member 108 and an active sensor member 122. The reference sensor member 108 may further comprise a reference conduit 110 including a reference conduit proximal end 140 defining a reference conduit first opening 114, a reference conduit distal end 142 defining a reference conduit second opening 116, and a reference conduit cavity 112 between said reference conduit proximal end 140 and said reference conduit distal end 142.

[0206] The reference conduit 110 can have a conduit wall 138 that can taper from a reference conduit proximal end 140 to a reference conduit distal end 142. In one embodiment, the reference conduit 110 can be substantially shaped as a cone or frustoconic with the reference conduit cavity 112 substantially shaped as a cone or frustoconic. In another embodiment, the reference conduit 110 can be substantially shaped as an elongated pyramid with a polygonally shaped base. For example, the reference conduit 110 can be substantially shaped as an elongated triangular pyramid, a square pyramid, or a pentagonal pyramid. In additional embodiments, the reference conduit 110 can be substantially shaped as a cylinder with a substantially cylindrical reference conduit cavity 112. In these embodiments, the reference conduit 110 can also have a tapered reference conduit distal end 142.

[0207] The reference conduit 110 can extend into the sample chamber 106 such that at least a portion of the reference conduit 110 is immersed in the sample 102 when the sample chamber 106 is filled with the sample 102. For example, the reference conduit distal end 142 can be immersed in the sample 102 in the sample chamber 106.

[0208] The reference sensor member 108 may include an ion exchange membrane 150 coupled to the reference conduit distal end 142. The ion exchange membrane 150 may be configured to block the reference conduit second opening 116 such that microorganisms in the sample 120 do not enter the reference conduit cavity 112. However, the ion exchange membrane 150 may allow ions to cross the ion exchange membrane 150 to maintain charge balance.

[0209] In some embodiments, the ion exchange membrane 150 can be a perfluorosulfonated ionomer membrane. As a more specific example, the ion exchange membrane 150 can be a Nafion® membrane provided by EI duPont de Nemours and Company. Also, for example, the ion exchange membrane 150 can be an Aciplex® membrane provided by Asahi Kasei Corporation or a Flemion® membrane provided by AGC Chemicals Americas, Inc.

[0210] In some embodiments, the ion exchange membrane 150 can have a thickness of about 25 μm to about 180 μm. More specifically, the ion exchange membrane 150 can have a thickness of about 25 μm to about 100 μm. The ion exchange membrane 150 can also have a thickness of about 100 μm to about 180 μm.

[0211] In another embodiment, the ion exchange membrane 150 can have a thickness of about 180 μm to about 200 μm. For example, the ion exchange membrane 150 can have a thickness of about 183 μm.

[0212] The reference conduit cavity 112 may be at least partially filled with a reference buffer 152. The reference buffer 152 may be an aqueous redox buffer.

[0213] In one embodiment, the reference buffer 152 can include deionized water, potassium ferricyanide, dihydrogen phosphate, disodium hydrogen phosphate, or a combination thereof. For example, the reference buffer 152 can include about 95% to about 99% (v / v%) deionized water, about 0.1% to 0.9% (w / v%) potassium ferricyanide, less than about 0.1% to 0.9% (w / v%) potassium ferricyanide, less than about 0.5% (w / v%) potassium dihydrogen phosphate, and less than about 0.5% (w / v%) disodium hydrogen phosphate. As a more specific example, the reference buffer can be 220 mV / pH 7 Redox Buffer (product code 51350060) provided by Mettler-Toredo AG.

[0214] In another embodiment, the reference buffer 152 can be an aqueous redox buffer containing 3 M KCl. As a more specific example, the reference buffer can be 3 M KCl Redox Buffer provided by Mettler-Toledo AG (Part No. 63056165).

[0215] In one embodiment, the reference conduit cavity 112 can be filled with about 100 μL of the reference buffer 152. In another embodiment, the reference conduit cavity 112 can be filled with about 500 μL of the reference buffer 152. In another embodiment, the reference conduit cavity 112 can be filled with about 100 μL to about 500 μL of the reference buffer 152. In a further embodiment, the reference conduit cavity 112 can be filled with about 500 μL to about 1 mL of the reference buffer 152. The amount of the reference buffer 152 can depend on the volume of the reference conduit cavity 112, the size of the reference conduit 110, the size of the sample vessel 104, or a combination thereof.

[0216] The reference sensor member 108 may also include a reference electrode 120 that extends into the reference conduit cavity 112 that is filled with the reference buffer solution 152. The reference electrode 120 may extend partially into the reference conduit cavity 112 that is filled with the reference buffer solution 152.

[0217] As a more specific example, the reference electrode 120 can include a reference electrode tip 144. The reference electrode tip 144 can be positioned proximate to the ion exchange membrane 150 where the reference electrode 120 extends partially into the reference conduit cavity 112 filled with a reference buffer 152. More specifically, a volume of the reference buffer 152 can separate the reference electrode tip 144 from the ion exchange membrane 150. The location of the reference electrode 120 within the reference conduit cavity 112 will be described in more detail in the next section.

[0218] The reference electrode 120 can be made in part of one or more metals. In one embodiment, the reference electrode 120 can be made in part of platinum (Pt), stainless steel, or a combination thereof. In another embodiment, the reference electrode 120 can be made in part of another type of conductive material. For example, the reference electrode 120 can be a metallic pin, wire, or rod inserted into the reference buffer 152 such that a segment of the reference electrode 120 (e.g., the reference electrode tip 144 and a segment of the reference electrode 120 proximate to the reference electrode tip 144) is immersed in the reference buffer 152.

[0219] The sample 102 can be in fluid contact with the ion exchange membrane 150 such that the ion exchange membrane 150 acts as the interface of the salt bridge. Ions can diffuse from one side of the ion exchange membrane 150 to the other to maintain charge balance.

[0220] The sensor device 105 may also include an active sensor member 122 including an active electrode housing 124 extending into a sample chamber 106 configured to receive a sample 102. The active sensor member 122 may also include an active electrode 148 at least partially contained or surrounded by the active electrode housing 124.

[0221] The active electrode housing 124 can be fabricated from an inert or non-conductive material and can include polymeric materials such as polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), ceramic, silicon dioxide (SiO2), or various types of glass, or combinations thereof.

[0222] The active electrode 148 can have a sample contacting surface 128. The active electrode 148 can be disposed at the active member distal end 130. The active electrode housing 124 can be configured to expose the sample contacting surface 128 such that the sample contacting surface 128 is in fluid communication with the sample 102.

[0223] In some embodiments, the active electrode 148 can be made in part of one or more metals, one or more metal oxides, or a combination thereof. For example, the active electrode 148 can be made in part of platinum (Pt), gold (Au), a redox-sensitive metal oxide, or a combination thereof. In some embodiments, the redox-sensitive metal oxide can include silicon dioxide (SiO2), aluminum oxide (Al2O3), titanium dioxide (TiO2), tantalum pentoxide (Ta2O5), hafnium dioxide (HfO2), iridium dioxide (IrO2), ruthenium dioxide (RuO2), zirconium dioxide (ZrO2), or a combination thereof.

[0224] The active electrode 148 and the reference electrode 120 may be electrically coupled to the voltmeter 136 by a conductive connection 134 or conductive wire. The conductive connection 134 may be made in part of a conductive material. In one embodiment, the conductive connection 134 may be made in part of a conductive material. In one embodiment, the conductive connection 134 may be a copper wire. For example, the conductive connection 134 may be electrodeposited copper, rolled annealed copper, high ductility electrodeposited copper, or a combination thereof. In another embodiment, the conductive connection 134 may be made in part of silver or nickel.

[0225] In some embodiments, the voltmeter 136 can be a high impedance voltmeter. The ORP of the sample 102 can be measured based on the potential difference between the active electrode 148 and the reference electrode 120.

[0226] The sensor device 105 may also include a vent conduit 146. A segment of the vent conduit 146 may extend into the sample chamber 106 such that a lumen or cavity within the vent conduit 146 is in fluid communication with the sample chamber 106. The vent conduit 146 may be configured to vent the sample 102 received within the sample container 104. Aeration of the sample 102 may increase the growth rate of microorganisms within the sample 102 by increasing the supply of oxygen to the microorganisms. Additionally, aeration may allow detachment of microorganisms from the inner walls of the sample chamber 104 to inhibit biofilm formation.

[0227] Figure 2 illustrates a top perspective view of one embodiment of sensor device 200. Sensor device 200 can be or refer to any of the sensors previously described, including sensor device 100 of Figure 1A, sensor device 101 of Figure 1B, sensor device 103 of Figure 1C, and sensor device 105 of Figure 1D.

[0228] 2, the sample vessel 104 can include a vessel cap 202 coupled to a vessel body 204. In one embodiment, the vessel body 204 can be a substantially cylindrical vessel or tube. In another embodiment, the vessel body 204 can be a substantially cubic, pyramidal, or conical vessel or tube. The vessel cap 202 can have a shape configured to couple or attach to the vessel body 204.

[0229] In one embodiment, the vessel cap 202 can be coupled to the sample vessel 104 by a threaded screw joint. The vessel cap 202 can be coupled to the sample vessel 104 by a catch, latch, interference fit, or combinations thereof. The vessel cap 202 can be removed or detached from the vessel body 203 to allow a user (e.g., a clinician or laboratory technician) to dispense the sample 102 into the sample chamber 106 of the sample vessel 104.

[0230] In one embodiment, the container cap 202 can have a reference port 206 defined along a surface of the container cap 202. The reference port 206 can be an opening or a channel defined on the surface of the container cap 202.

[0231] In some embodiments, the reference conduit 110 can be integrated with the vessel cap 202 such that separation of the vessel cap 202 from the vessel body 204 also removes the reference conduit 110 from the sample chamber 106. In these and other embodiments, the reference port 206 can be an opening or channel that provides access to the reference conduit cavity 112. For example, the reference electrode 120 can be inserted or otherwise extended into the reference conduit cavity 112 through the reference port 206 and the reference conduit first opening 114.

[0232] In another embodiment, the entire reference sensor member 108, including the reference conduit 110, can be removably coupled to the sample vessel 104 such that the entire reference sensor member 108 is detachable from the sample vessel 104. For example, the reference conduit 110 can be removably coupled to the vessel cap 202. In these and other embodiments, the reference port 206 can provide access to the sample chamber 106. The reference conduit 110 (including either the reference buffer gel 118 or the reference buffer 152) can be inserted or otherwise extended from the reference port 206.

[0233] 2 shows that the container cap 202 can also have a vent 208 defined along a surface of the container cap 202. The vent 208 can provide access to the vent conduit 146 (see any of FIGS. 1A, 1B, 1C, or 1D). For example, a vent tube or hose can be inserted from the vent 208 into the vent conduit 146 to deliver air to the sample 102 in the sample container 104.

[0234] The vessel cap 202 may also have an exhaust vent 210 defined along a surface of the vessel cap 202. The exhaust vent 210 may allow excess air or gaseous by-products produced by the microorganisms within the sample vessel 104 to escape from the sample chamber 106.

[0235] The sample vessel 104 may also include a side port 212 defined along a side of the vessel body 204. The side port 212 may be an opening or channel that provides access to the sample chamber 106. At least a portion of the active sensor member 122 may extend from the side port 212 when coupled to the sample vessel 104. In some embodiments, the active sensor member 122 may also be removably coupled to the sample vessel 104 such that a new instance of the active sensor member 122 may replace an older or used instance of the active sensor member 122.

[0236] Figure 3A shows a side cross-sectional view of one embodiment of a reference sensor member 108. The reference sensor member 108 shown in Figure 3A can be used as part of the sensor device 100 of Figure 1A or the sensor device 101 of Figure 1B.

[0237] The reference conduit 110 can have a conduit wall 138 that tapers from a reference conduit proximal end 140 to a reference conduit distal end 142. Additionally, the reference conduit proximal end 140 can define a first open area that is greater than a second open area defined by the reference conduit distal end 142. For example, the first open area can be about 19 mm 2 ~ approx. 80 mm 2and the second opening area is about 0.79 mm 2 ~ approx. 3.14 mm 2 It can be.

[0238] In the exemplary embodiment shown in Figure 3A, the reference conduit 110 can be substantially shaped as an elongated inverted cone. In this embodiment, both the reference conduit first opening 113 and the reference conduit second opening 116 can be substantially circular and defined by a first opening diameter 300 and a second opening diameter 302, respectively. In some cases, the first opening diameter 300 can be between about 5.00 mm and about 10.0 mm. The second opening diameter 302 can be between about 1.00 mm and about 5.00 mm.

[0239] FIG. 3A also shows that the reference buffer gel 118 can be filled to a gel height 304 of about 5.00 mm to about 20.0 mm. The gel height 304 can vary depending on the volume of the reference conduit cavity 112. As previously described, the reference conduit cavity 112 can be filled with about 100 μL of the reference buffer gel 118. In another embodiment, the reference conduit cavity 112 can be filled with about 500 μL of the reference buffer gel 118. In other embodiments, the reference conduit cavity 112 can be filled with about 100 μL to about 500 μL of the reference buffer gel 118. In further embodiments, the reference conduit cavity 112 can be filled with about 500 μL to about 1 mL of the reference buffer gel 118. The amount of the reference buffer gel 118 can be optimized to achieve a predetermined reference time. For example, in some cases, the amount of reference buffer gel 118 can be optimized to achieve a reference time between 3.0 hours and 6.0 hours.

[0240] The reference electrode 120 may also be separated from the reference conduit second opening 116 by a separation distance 306. In one embodiment, the separation distance may be about 1.0 mm. In another embodiment, the separation distance 306 may be about 5.0 mm. In another embodiment, the separation distance 306 may be between about 1.0 mm and about 5.0 mm.

[0241] Figure 3B shows a side cross-sectional view of another embodiment of a reference sensor member 108. The reference sensor member 108 shown in Figure 3B can be used as part of the sensor device 103 of Figure 1C or the sensor device 105 of Figure 1D.

[0242] The reference conduit 110 can include a conduit wall 138 that is tapered from a reference conduit proximal end 140 to a reference conduit distal end 142. Additionally, the reference conduit proximal end 140 can define a first open area that is larger than a second open area defined by the reference conduit distal end 142. For example, the first open area can be about 19 mm 2 ~ approx. 80 mm 2 and the second opening area can be about 0.79 mm 2 ~ approx. 3.14 mm 2 It can be.

[0243] In the exemplary embodiment of FIG. 3B, the reference conduit 110 can be substantially shaped as an elongated upside-down frustoconic. In this embodiment, both the reference conduit first opening 114 and the reference conduit second opening 116 can be substantially circular and defined by a first opening diameter 300 and a second opening diameter 302, respectively. In some cases, the first opening diameter 300 can be between about 5.00 mm and about 10.0 mm. The second opening diameter 302 can be between about 1.00 mm and about 5.00 mm.

[0244] FIG. 3B also shows that the reference buffer 152 can be filled to a solution height 308 of about 5.00 mm to about 20.0 mm. The solution height 308 can vary depending on the volume of the reference conduit cavity 112. As previously described, the reference conduit cavity 112 can be filled with about 500 μL of the reference buffer 152. In another embodiment, the reference conduit cavity 112 can be filled with about 100 μL to about 500 μL of the reference buffer 152. In a further embodiment, the reference conduit cavity 112 can be filled with about 500 μL to about 1 mL of the reference buffer 152. The amount of the reference buffer 152 can be optimized to achieve a predetermined reference time. For example, in some cases, the amount of the reference buffer 152 can be optimized to achieve a reference time between 3.0 hours and 6.0 hours.

[0245] The reference electrode 120 may also be separated from the ion exchange membrane 150 by a separation distance 306. In one embodiment, the separation distance may be about 1.0 mm. In another embodiment, the separation distance 306 may be about 5.0 mm. In another embodiment, the separation distance 306 may be between about 1.0 mm and about 5.0 mm.

[0246] The ion exchange membrane 150 can have a thickness 310 of about 25 μm to about 180 μm. More specifically, the ion exchange membrane 150 can have a thickness 310 of about 25 μm to about 100 μm. The ion exchange membrane 150 can also have a thickness 310 of about 100 μm to about 180 μm.

[0247] 4 shows a schematic diagram of an active electrode (e.g., active electrode 126 or active electrode 148) and a reference electrode (e.g., reference electrode 120) electrically coupled to a high impedance voltmeter (e.g., voltmeter 136). As previously described, the reference sensor member 108 can be configured such that either the reference buffer gel 118 or the ion exchange membrane 150 is in fluid contact (or liquid-solid contact) with the sample 102 in the sample container 104. In addition, the active electrode (e.g., active electrode 126 or active electrode 148) can also be in fluid contact with the sample 102 in the sample container 104.

[0248] The reference electrode can be immersed or inserted into either the reference buffer gel 118 or the reference buffer solution 152 and electrically coupled to a high impedance voltmeter (e.g., voltmeter 136). The active electrode can also be electrically coupled to the high impedance voltmeter via a conductive connection 134. The reference electrode can be hung at a stable half-cell potential compared to the active electrode, and the potential difference between the active and reference electrodes can be used to determine the pH or ORP of the sample 102.

[0249] 5A is a graph showing ORP measurements made using a conventional KCl reference solution and a stainless steel reference electrode in a reference buffer gel after the addition of a high potential ORP solution. To test the effectiveness of the sensor device 101 (see FIG. 1B), a known high potential ORP solution (e.g., Virkon TM A solution of 100 mg of 100% EDTA (aqueous mixture containing EDTA powder) was added to an ORP buffer (e.g., 220 mV / pH 7 ORP buffer). The resulting change in potential difference was measured using a conventional ORP probe (e.g., Mettler Toledo probe provided by Mettler-Toledo AG) with a conventional KCl reference solution. TM Measurements were made using both the conventional ORP probe (K.S. Patent No. 5,313,666) and a sensor device 101 with a stainless steel reference electrode 120 immersed in a reference buffer gel 118. The conventional ORP probe and the sensor device 101 used the same platinum (Pt) active electrode. As shown in FIG. 5A, both sensor setups behaved similarly in response to the addition of known high potential ORP solutions. The reference sensor element 108 with the reference buffer gel 118 maintained a stable reference potential similar to the conventional KCl reference in the conventional ORP probe.

[0250] FIG. 5B is a graph showing pH measurements made using a conventional KCl reference solution and a platinum reference electrode in a reference buffer gel after replacing a pH 7 solution with a pH 4 solution. To evaluate the effectiveness of the sensor device 100 (see FIG. 1A), a pH change was induced by replacing the pH 7 solution with a pH 4 solution. The resulting change in potential difference was measured using a conventional pH probe (e.g., a platinum reference electrode provided by Mettler-Toledo AG) using a conventional KCl reference solution. TM Measurements were performed using both a conventional pH probe (Trademark) and a sensor device 100 with a platinum reference electrode 120 immersed in a reference buffer gel 118. The conventional pH probe and the sensor device 100 used the same iridium dioxide (IrO2) active electrode. As shown in FIG. 5B, both sensor setups performed similarly in response to a step change in pH. The reference sensor element 108 with the reference buffer gel 118 exhibited similar sensitivity and step height as the conventional pH probe with a KCl reference solution. A reference potential was maintained.

[0251] FIG. 6 is a graph showing ORP measurements of E. coli bacterial growth using a reference electrode in 1% agar gel buffer, 5% agar gel buffer, and a conventional KCl reference solution. As shown in FIG. 6, the ORP of a solution containing E. coli was monitored using a conventional ORP probe with a conventional KCl reference solution and two additional instances of the sensor device 101. In the first instance, the reference buffer gel 118 contained 1% (w / v%) agar powder. In the second instance, the reference buffer gel 118 contained 5% (w / v%) agar powder. The same platinum (Pt) active electrode was used for all three sensor configurations. As shown in FIG. 6, all three sensor configurations performed similarly and produced similar growth curves. All differences in signal timing were within the variability range of the three platinum (Pt) active electrodes.

[0252] FIG. 7A shows the comparison of a conventional ORP buffer with a high potential ORP solution (e.g., Virkon TMFIG. 1 is a graph showing constant ORP measurements taken when the conventional ORP buffer was replaced with the high potential ORP solution and then switched back to the conventional ORP buffer. The dashed line shows the ORP signal measured using a conventional ORP probe with a conventional KCl reference solution. As expected, the ORP signal measured by the ORP probe rose sharply when the conventional ORP buffer was replaced with the high potential ORP solution, and then fell again when the high potential ORP solution was switched back to the conventional ORP buffer.

[0253] The solid line shows a conventional reference sensor element containing a conventional KCl reference solution and an ion exchange membrane 150 (e.g., Nafion TM 7A shows the measurement of the potential difference between the conventional reference sensor element and the reference sensor element containing the ion exchange membrane 150. As shown in FIG. 7A, the potential difference between the conventional reference sensor element and the reference sensor element containing the ion exchange membrane 150 remained unchanged even when the ORP reference solution was exchanged for the high potential ORP solution.

[0254] FIG. 7B is a graph showing the ORP measurement of Escherichia coli (E. coli) bacterial growth using a conventional ORP probe and the potential difference between a reference sensor element having an ion exchange membrane and a conventional reference sensor element having a KCl reference solution. As shown in FIG. 7B, the ORP of a solution containing E. coli was monitored using a conventional ORP probe with a conventional KCl reference solution. The dashed line in FIG. 7B shows the result of such a measurement. The solid line in FIG. 7B shows the potential difference between the conventional KCl reference solution and the reference sensor element containing the ion exchange membrane 150. As shown in FIG. 7B, the potential difference remained unchanged throughout the E. coli growth period (up to 15 hours).

[0255] 8 illustrates another embodiment of a sensor apparatus 800 for measuring a solution property (e.g., pH or ORP) of a sample 102. The sensor apparatus 800 includes a sample container 802 having a sample chamber 908 (e.g., FIG. 9A ) defined therein. The sample chamber 908 can be configured to receive the sample 102. The sensor apparatus 800 can also include a reference sensor member 804. In some embodiments, the reference sensor member 804 can be fabricated as part of a container cap 806 configured to cover an open end of the sample container 802.

[0256] In some embodiments, the sample vessel 802 can be a cuvette. In other embodiments, the sample vessel 802 can be a diagnostic tube, a portion of a test tube, a glass vessel, or another type of laboratory vessel or tube.

[0257] In some embodiments contemplated by this disclosure, the sample vessels 802 may refer to sample wells of a well plate. The well plate may be a general-purpose well plate having deeper wells than a conventional well plate or a microtiter plate.

[0258] The reference sensor member 804 can comprise a reference conduit 900 including a reference conduit cavity 902, a reference conduit first opening 904 and a reference conduit second opening 906 (see, e.g., FIGS. 9A and 9B). The reference conduit 900 can be an elongated channel or passage configured to extend into a sample chamber 908 of the sample vessel 802.

[0259] The sensor apparatus 800 may also include a wicking member 910 disposed within and extending along the length of the reference conduit cavity 902 (see, e.g., FIGS. 9A and 9B). The wicking member 910 and the reference conduit cavity 902 will be discussed in more detail in the next section.

[0260] The sensor device 800 can further include a reference electrode material 808 disposed at a proximal end of the wicking member 910. If the reference sensor member 804 is fabricated as a container cap 806, the reference electrode material 808 can protrude or extend from a cap tip 810 of the container cap 806 such that the reference electrode material 808 is accessible and visible from the cap tip 810 of the container cap 806. For example, an opening can be defined along the cap tip 810 of the container cap 806 and the reference electrode material 808 can protrude or extend from the opening defined along the cap tip 810 of the container cap 806. In another embodiment not shown in the drawings but contemplated by the present disclosure, the reference electrode material 808 can protrude from a side 812 or corner of the container cap 806.

[0261] If the reference sensor member 804 is fabricated as a vessel cap 806, the vessel cap 806 may be removably attached or coupled to the sample vessel 802. For example, the vessel cap 806 may be removably attached or coupled to the sample vessel 802 via a threaded joint 944 (see FIGS. 9A and 9B). In another embodiment, the vessel cap 806 may be removably attached or coupled to the sample vessel 802 via an interference fit, a press fit (e.g., a tapered press fit, an expansion press fit, etc.), a snap fit, a latch, or a combination thereof.

[0262] In these or other embodiments, the vessel cap 806 may fit over or cover a portion (e.g., a proximal portion) of the sample vessel 802. In these embodiments, the side 812 of the vessel cap 806 may extend radially beyond the vessel wall 814 of the sample vessel 802. The sample vessel 802 may be substantially cylindrical, with the shape of the vessel cap 806 conforming to the shape of the sample vessel 802, and the circumference (or circular footprint) of the vessel cap 806 being larger than the circumference (or circular footprint) of the sample vessel 802.

[0263] The sensor device 800 may also include an active sensor element 816. The active sensor element 816 may be set or otherwise disposed along a vessel wall 814 of the sample vessel 802. The active sensor element 816 will be discussed in more detail in the next section. The reference sensor element 804 and the active sensor element 816 may be electrically coupled to a parameter analyzer 916 by a conductive connection (e.g., FIG. 9A). A solution property of the sample 102 may be determined based on a potential difference measured between an active electrode material 918 (e.g., FIG. 9A) of the active sensor element 816 and the reference electrode material 808.

[0264] In some embodiments, the solution property measured or monitored using the sensor device 800 can be the pH of the sample 102, and at least a portion of the active sensor member 816 can be made of a pH sensitive material. The pH sensitive material can include at least one of silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium dioxide, ruthenium dioxide, and zirconium dioxide.

[0265] In these and other embodiments, the solution property measured or monitored using the sensor device 800 can be an oxidation-reduction electrode (ORP) of the sample 102, and at least a portion of the active sensor member 816 can be made of a portion of a redox-sensitive material. The redox-sensitive material can include platinum, gold, silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium dioxide, ruthenium dioxide, and zirconium dioxide.

[0266] As discussed above, the sample 102 may contain susceptible pathogens or microorganisms (e.g., bacteria, fungi, etc.). The sensor device 800 may be used to analyze the sample 102 for the growth or lack thereof of microorganisms or susceptible pathogens as part of a microbial quantification method, a sample preparation method, or an antibiotic susceptibility testing (AST) method.

[0267] As discussed above, sample 102 may include or refer to a bacterial culture derived from at least one of a sample obtained from a patient or subject, a biological sample, an environmental sample, and a food sample. For example, sample 102 may include or refer to a bacterial culture or a resuspended bacterial culture derived from a bodily fluid or specimen (swab) obtained from a patient or subject. As a more specific example, sample 102 may include a bacterial culture or a resuspended bacterial culture that has tested positive for microbial growth.

[0268] More specifically, sample 102 may include a bacterial culture derived from blood obtained from a patient or subject that has tested positive for microbial growth. In some embodiments, sample 102 may be or may be referred to as a positive blood culture. For purposes of this disclosure, a positive blood culture may be a bacterial culture derived from blood taken from a patient or subject that has tested positive for bacterial growth.

[0269] In some embodiments, the aqueous growth medium can be added to the sample 102 to dilute the sample 102 prior to introduction into the sample vessel 802. In other embodiments, the aqueous growth medium can be added to the sample 102 after the sample 102 has been injected, delivered or otherwise introduced into the sample vessel 802.

[0270] In one embodiment, the aqueous growth medium can be Muller Hinton Broth (MHG) supplemented with glucose, in another embodiment, the aqueous growth medium can be Bactotryptone, tryptic soy digest, yeast extract, beef extract, cation adjusted Muller Hinton Broth (CAMHB), starch, acid hydrolysate of casein, calcium chloride, magnesium chloride, sodium chloride, blood or hemolysate including horse hemolysate (LHB), CAMHB-LHB, glucose or other carbohydrates, or combinations thereof.

[0271] The microorganism or infectious agent that can be assayed using the sensor device 800 can be any metabolic unicellular or multicellular organism, including any of the bacterial and fungal species or genera mentioned above.

[0272] The sample container 802, container cap 806, or combination thereof, can be fabricated in part from a non-conductive material. The sample container 802, container cap 806, or combination thereof, can be fabricated in part from a non-leaching or sterilizable material. The sample container 802, container cap 806, or combination thereof, can be fabricated in part from a polymeric material (e.g., a thermoplastic, a photocurable polymer, or a combination thereof), a ceramic material, or glass, or a combination thereof. For example, the sample container 802, container cap 806, or combination thereof, can be fabricated in part from polycarbonate, polypropylene (PP), nylon, acrylonitrile-butadiene-styrene (ABS), polyethylene (PE), polystyrene (PS), polyvinyl chloride (PVC), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), or a combination thereof. As a more specific example, the sample container 802, container cap 806, or a combination thereof, may be made in part from molded polycarbonate or a cured low viscosity liquid photopolymer such as Somos® WaterShed XC.

[0273] 9A illustrates a side cross-sectional view of a sensor device 800. As shown in FIG 9A, the sensor device 800 can include a reference sensor member 804 fabricated as a vessel cap 806 and a sample vessel 802. The vessel cap 806 can be removably or detachably coupled or secured to the sample vessel 802.

[0274] The reference sensor member 804 may comprise a reference conduit 900 having a reference conduit cavity 902, a reference conduit first opening 904, and a reference conduit second opening 906. The reference conduit 900 may be an elongated channel or passage configured to extend into a sample chamber 908 of the sample vessel 802.

[0275] The sensor apparatus 800 may also include a wicking member 910 disposed within or extending along the length of the reference conduit cavity 902. In some embodiments, the wicking member 910 may fill or occupy all of the space within the reference conduit cavity 902. In other embodiments, the wicking member 910 may partially fill or occupy the space within the reference conduit cavity 902.

[0276] The wicking member 910 can have a wick distal end 912 and a wick proximal end 914. At least a portion of the wicking member 910 can be in fluid communication with the sample chamber 908 such that when the reference sensor member 804 (e.g., vessel cap 806) is attached, secured or coupled to the sample vessel 802, at least a substantial amount of the sample in the sample chamber 908 is drawn up, absorbed or otherwise wicked by at least a portion of the wick distal end 912 toward the wick proximal end 914.

[0277] In some embodiments, at least a portion of the wick distal end 912 can extend through the second opening 906 of the reference conduit such that the wick distal end 912 protrudes or extends into the sample chamber 908. In these embodiments, the wick distal end 912 extends or protrudes into the liquid sample 102 when the sample chamber 908 is filled with sample 102.

[0278] In another embodiment, the entire wick distal end 912 is disposed proximal or above the reference conduit second opening 906 such that the wick distal end 912 does not protrude or extend into the sample chamber 908. In these embodiments, the wick distal end 912 is still in fluid communication with the sample chamber 908, and the liquid sample 102 can reach or contact the wick distal end 912 by being drawn up into the reference conduit 900 by capillary action or by perturbing / shaking the sample vessel 802.

[0279] 9A illustrates that the reference conduit 900 may be tapered such that the volume of the reference conduit cavity 902 tapers or narrows from the reference conduit first opening 904 to the reference conduit second opening 906. The shape of the wicking member 910 may match or conform to the shape of the reference conduit cavity 902. The wicking member 910 may be configured such that the shape of the wicking member 910 tapers or narrows from the wick proximal end 914 to the wick distal end 912.

[0280] The wicking member 910 may be made in part from a porous material. The wicking member 910 may be made in part from a material that includes pores sized to about 15 μm to about 150 μm (eg, about 50 μm).

[0281] In some embodiments, the wicking member 910 can be made in part from a polymeric material. As a more specific example, the wicking member 910 can be made in part from a porous polymeric material that includes pores sized to between 15 μm and about 150 μm. In one embodiment, the wicking member 910 can be made in part from high density polyethylene (HDPE). For example, the wicking member 910 can be made in part from HDPE with pores sized to about 50 μm.

[0282] In another embodiment, the wicking member 910 may be made in part from natural fibers. For example, the wicking member 910 may be made in part from cellulosic fibers, pulp, paper, cotton, or combinations thereof.

[0283] The wicking component 910 may be treated with a surfactant such that at least the surface of the wicking component 910 is covered with the surfactant. In some embodiments, the wicking component 910 may be treated with a surfactant or saturated by immersion in a solution containing the surfactant prior to introduction into the reference conduit 902. The surfactant may be configured to increase the hydrophilicity of the wicking component 910 (i.e., to render a substantially hydrophobic surface of the wicking component 910 more hydrophilic). In some embodiments, the surfactant may be a fluorosurfactant. In another embodiment, the surfactant may be a non-ionic surfactant such as one or more poloxamers. As a more specific example, the surfactant may include Pluronic® F-68.

[0284] The reference sensor member 804 may also include a reference electrode material 808 disposed at the wick proximal end 914. The reference electrode material 808 may be a conductive material provided or dispensed on the wick proximal end 914.

[0285] In some embodiments, the reference electrode material 808 may be a conductive ink provided or dispensed on the wick proximal end 914. The conductive ink provided or dispensed on the wick proximal end 914 may be hardened by curing. More specifically, the conductive ink may be a silver-silver chloride (Ag-AgCl) ink.

[0286] At least a portion of the reference electrode material 808 can be in physical contact with the wicking member 910. In some embodiments, at least a portion of the reference electrode material 808 can protrude or extend beyond the container cap 806. The reference electrode material 808 can be a cured material disposed at the wick proximal end 914. In some embodiments, the reference electrode material 808 can be disposed in the center of the container cap 806.

[0287] One advantage of the wicking member 910 disclosed herein is that the wicking member 910 can draw up the liquid sample 102 and allow the sample 102 to wick through the pores of the wicking member 910 towards the reference electrode material 808. For example, the liquid sample 102 can be wicked towards the wick proximal end 914 where it is in fluid contact with the reference electrode material 808.

[0288] When the reference electrode material 808 is made of a material such as silver-silver chloride (Ag-AgCl), the wicking member 910 traps silver ions (Ag) that would otherwise freely diffuse into the sample 102. + ) that may be harmful or otherwise affect the growth of microorganisms or infectious agents in the sample 102. The wicking member 910 may act as a barrier or obstacle to the harmful silver ions by slowing or stalling the diffusion of such ions into the sample 102. A wicking member 910 having the dimensions and shapes disclosed herein may be effective in slowing or stalling the diffusion of such harmful ions.

[0289] The reference sensor member 804 can be electrically coupled to a parameter analyzer 916 by a conductive connection 134. The parameter analyzer 916 can be a voltmeter or a multimeter. For example, the parameter analyzer 916 can be a high impedance voltmeter, such as the voltmeter 136 described above.

[0290] The sensor apparatus 800 may also include an active sensor element 816 mounted or otherwise disposed along a vessel wall 814 of the sample vessel 802. In another embodiment not shown in the drawings, the active sensor element 816 may be mounted or otherwise disposed along the bottom of the sample vessel 802.

[0291] The active sensor member 816 may include an active electrode material 918, an active electrode housing 920, and a conductive contact layer 922. At least a portion of the active electrode material 918 may be in fluid communication with the sample chamber 908. For example, at least a portion of the active electrode material 918 may extend or protrude into the sample chamber 908. The active electrode material 918 may be positioned such that at least a layer or surface of the active electrode material 918 is in fluid contact with the sample 102 when the sample chamber 908 is at least partially filled with the sample 102.

[0292] The active electrode housing 920 can partially surround or contain the active electrode material 918, the conductive contact layer 922, or a combination thereof. The active electrode housing 920 is made of the same material as the vessel wall 814 and is a molded feature of the vessel wall 814. In some embodiments, the active electrode housing 920 can be a conduit or channel defined along the vessel wall 814 of the sample vessel 802. In another embodiment, the active electrode housing 920 is defined along the bottom of the sample vessel 802. The active electrode housing 920 can expose at least a portion of the active electrode material 918 at one end of the active electrode housing 920 and at least a portion of the conductive contact layer 922 at the other end of the active electrode housing 920. In certain embodiments, one or more conductive connections 134 can be in physical contact with the conductive contact layer 922 or the active electrode material 918 through the active electrode housing 920.

[0293] The conductive contact layer 922 may be coupled to the active electrode material 918 and may be located proximate or radially outwardly of the active electrode material or otherwise disposed therein (e.g., close toward the outside of the sample container 802). The conductive contact layer 922 may be made in part of aluminum (Al), copper (Cu), platinum (Pt), or combinations thereof. The conductive contact layer 922 may be electrically coupled to the reference electrode material 808 by the conductive connection 134 via the parameter analyzer 916. The conductive connection 134 may be made in part of a conductor material. The conductive connection 134 may be a conductor wire or conductive line. In one embodiment, the conductive connection 134 may be a copper wire or copper trace. For example, the conductive connection 134 may be electrodeposited copper, rolled annealed copper, highly ductile electrodeposited copper, or combinations thereof. In other embodiments, the conductive connection 134 may be made in part of silver or nickel. The conductive connection 134 may be in contact with the reference sensor member 804 and the active sensor member 816 when the sample container 802 is at least partially filled with the sample 102 .

[0294] The solution properties of the sample 102 can be determined based on the potential difference measured between the active electrode material 918 and the reference electrode material 808. The reference electrode material 808 can function as a reference electrode and provide a stable half-cell potential compared to the active electrode material 918, and the potential difference between the active electrode and the reference electrode can be used to determine the pH or ORP of the sample. For example, the reference electrode material 808 (e.g., an Ag-AgCl ink) can be configured to exhibit a substantially stable electrode potential compared to the active electrode material 918 when the active electrode material 918 is in electrical communication with the reference electrode material 808.

[0295] In some embodiments, the solution property measured or monitored can be the pH of the sample 102. In another embodiment, the solution property measured or monitored can be the oxidation-reduction potential (ORP) of the sample 102.

[0296] If the solution property being measured or monitored is pH, the active electrode material 918 can be a pH sensitive material. The pH sensitive material can be or include any of silicon dioxide (SiO2), aluminum oxide (Al2O3), titanium dioxide (TiO2), tantalum pentoxide (Ta2O5), hafnium dioxide (HfO2), iridium dioxide (IrO2), ruthenium dioxide (RuO2), zirconium dioxide (ZrO2), or combinations thereof.

[0297] When the solution property being measured or monitored is ORP, the active electrode material 918 can be a redox sensitive material. The redox sensitive material can be or include either platinum (Pt), gold (Au), a redox sensitive metal oxide, or a combination thereof. More specifically, the redox sensitive material can be or include either silicon dioxide (SiO2), aluminum oxide (Al2O3), titanium dioxide (TiO2), tantalum pentoxide (Ta2O5), hafnium dioxide (HfO2), iridium dioxide (IrO2), ruthenium dioxide (RuO2), zirconium dioxide (ZrO2), or a combination thereof.

[0298] 9A, when the reference sensor member 804 is implemented as a container cap 806, the container cap 806 can have dimensions defined by a cap width 924 (or radius) and a bottom cap height 926. In some embodiments, the cap width 924 can be about 10.0 mm to about 20.0 mm. For example, the cap width 924 can be about 15.7 mm. In some embodiments, the cap height 926 can be about 5.0 mm to about 20.0 mm. For example, the cap height 926 can be about 10.5 mm. When the container cap 806 is fastened, attached, or otherwise coupled to the sample container 802, the sensor device 800 can have a device height 928 measured from the bottom of the sample container 802 to the cap top 810 of the container cap 806.

[0299] In some embodiments, device height 928 can be from about 20.0 mm to about 50.0 mm. In other embodiments, device height 928 can be from about 25.0 mm to about 35.0 mm. For example, device height 928 can be about 31.3 mm.

[0300] 9A, the wicking member 910 can have a wick height 930 as measured from the wick proximal end 914 to the wick distal end 912. In some embodiments, the wick height 930 can be between about 10.0 mm and about 20.0 mm. More specifically, the wick height 930 can be between about 14.0 mm and about 15.0 mm. For example, the wick height 930 can be about 14.8 mm.

[0301] One advantage of a wicking member 910 having a wick height 930 is that such a wicking member 910 allows a sufficient amount of sample to be drawn up toward the reference electrode material 808 while acting as a barrier or obstacle to certain harmful ions (e.g., silver ions) flowing from the reference electrode material 808 toward the microorganism-rich sample 102 in the sample chamber 908.

[0302] 9A also shows that the reference electrode material 808 has a reference material height 932 (or thickness) and a reference material width 934 (or diameter). When the reference electrode material is a cured and hardened mass of silver-silver chloride (Ag-AgCl), the reference electrode material height 932 and the reference material width 934 can be the height and width, respectively, of the cured mass of silver-silver chloride.

[0303] In some embodiments, the height 932 of the reference material can be between about 0.2 mm and 1.0 mm. For example, the height 932 of the reference material can be about 0.4 mm. In some embodiments, the width 934 of the reference material can be between about 2.0 mm and about 5.0 mm. For example, the width 934 of the reference material can be about 3.0 mm. One advantage of the reference electrode material 808 disclosed herein is that the reference electrode material 808 can function as a stable reference electrode, i.e., can provide a stable reference potential for up to 10 hours of testing or operation.

[0304] 9A, the reference electrode material 808 can be at least partially located or disposed within a divot, depression or recessed portion created by the wicking member 910 and the reference conduit 900. When the reference electrode material 808 is a cured or hardened conductive ink or solution (e.g., Ag-AgCl ink), the divot, depression or recessed portion can act as a receiving space for the liquid ink or solution to be cured.

[0305] FIG. 9A also shows that the sensor device 800 can include a vent 936 defined along the bottom of the sample container 802, the container wall 814 of the sample container 802, or a combination thereof. The vent 936 can be covered by a first breathable membrane 938. The vent 936 and the first breathable membrane 938 can be configured to allow gas 940 to enter the sample chamber 908. In some embodiments, the gas 940 can be ambient air (e.g., air in a laboratory, clinical facility, or testing facility). In another embodiment, the gas 940 can include a combination of pressurized oxygen, carbon dioxide, nitrogen, and argon. The vent 936 and the first breathable membrane 938 allow air to enter the sample chamber 908 through the vent 936 and aerate the sample 102 in the sample chamber 908. Aeration of the sample 102 may favor aerobic organisms, such as Pseudomonas aeruginosa or Acinetobacter baumanii, by providing an oxygen-rich environment within the sample chamber 908. Aeration of the sample 102 may accelerate the growth of the microbial population within the sample 102.

[0306] The vent 936 may be an opening or channel defined along the bottom of the sample vessel 802, the vessel wall 814 of the sample vessel 802, or a combination thereof. For example, the vent 936 may be defined in the center or centre of the vessel bottom.

[0307] In a further embodiment, a vent 936 can be defined along the cap apex 810 of the vessel cap 806 and a gas 940 can be pumped into the sample chamber 908 from the top of the sample vessel 802 .

[0308] Gas 940 (e.g., ambient air) can be pumped into sample chamber 908 by a micropump or another pump type device. Gas 940 (e.g., ambient air) can be pumped or otherwise provided through vent 936 and first breathable membrane 938 into sample chamber 908 at a constant flow rate of about 1.0-10.0 mL / min. In another embodiment, gas 940 (e.g., ambient air) can be pumped or otherwise directed through vent 936 and first breathable membrane 938 into sample chamber 908 at a particular duty cycle.

[0309] 9A also shows that a second breathable membrane 942 can cover at least a portion of the bottom surface of the container cap 806. The second breathable membrane 942 can allow any gas 940 to be pumped or otherwise introduced into the sample chamber 908 to exit the sample chamber while preventing any liquid within the sample chamber 908 from leaking out of the sample container 802. The second breathable membrane 942 also allows any gaseous by-products generated or generated within the sample chamber to exit the sample chamber 908.

[0310] In some embodiments, the first breathable membrane 938 and the second breathable membrane 942 are manufactured from the same material. The first breathable membrane 938 and the second breathable membrane 942 can be made of a hydrophobic breathable film or thin sheet. For example, the first breathable membrane 938 and the second breathable membrane 942 can both be made of or include polytetrafluoroethylene (PTFE).

[0311] 9A, the vessel cap 806 can be removably or detachably coupled or fastened onto a proximal portion of the sample vessel 802 via a threaded joint 944. When the vessel cap 806 (which functions as part of the reference sensor member 804) is fastened or coupled to the sample vessel 802 by the threaded joint 944, an air flow path can be established such that air can pass through the first breathable membrane 938, into the vent 936, into the sample chamber 908, and out of the sample chamber 908 through the second breathable membrane 942, defining an air gap 948 between the vessel cap 806 and the sample vessel 802.

[0312] 9B illustrates one embodiment of the reference sensor member 804 of the sensor apparatus 800. As shown in FIG. 9B, the reference sensor member 804 can be implemented or manufactured as a container cap 806 including a reference conduit 900 extending from a bottom surface 950 of the container cap 806. The reference conduit 900 can be an elongated channel extending from the bottom surface 950 of the container cap 806. The reference conduit 900 can be hollow and can be defined by a reference conduit cavity 902 extending from a reference conduit first opening 904 to a reference conduit second opening 906.

[0313] The reference conduit cavity 902 can extend through the entire length of the reference conduit 900. The reference conduit cavity 902 can be shaped as a substantially cylinder, an elongated cone, an elongated fructoconic, an elongated rectangular solid, or combinations thereof. The shape of the wicking member 910 can fit and match the shape of the reference conduit cavity 902.

[0314] The container cap 806 can be made in part of a transparent or clear material or a transparent or clear non-conductive material. In other embodiments, the container cap 806 can be made in part of a translucent or see-through material. For example, as shown in FIG. 9B, at least a portion of the wicking member 910 is visible through a side 812 of the container cap 806. This allows a user or operator of the sensor device 800 to observe the wicking phenomenon of the fluid sample 102 from the wick distal end 912 to the wick proximal end 914 when the container cap 806 is secured to the sample container 802, ensuring that at least a portion of the sample 102 can reach the reference electrode material 808 at the wick proximal end 914. In some embodiments, the container cap 806 can be made in part of a transparent or clear polymeric material, glass, or a combination thereof.

[0315] The method of manufacturing the reference sensor member 804 includes providing a vessel cap 806 configured to be removably coupled to a sample vessel 802. The sample vessel 802 can be configured to receive a sample 102. As described above, the reference conduit 900 can extend from a bottom surface 950 of the vessel cap 806. The reference conduit 900 can include a reference conduit cavity 902. In these and other embodiments, the method also includes casting the reference sensor member 804 in the shape of the vessel cap 806 with the reference conduit 900 defined in the center of the vessel cap 806. For example, the vessel cap 806 can be molded through injection molding or casting. In another embodiment, the method can include 3D printing the vessel cap 806 with the reference conduit 900 defined in the center of the vessel cap 806 and the reference conduit cavity 902 extending the length of the reference conduit 900.

[0316] The method may further include installing or disposing a wicking member 910 in the reference conduit cavity 902 of the reference conduit 900. The wicking member 910 may include a wick distal end 912 and a wick proximal end 914. For example, the wicking member 910 may be made in part from a porous polymeric material (e.g., HDPE), natural fibers, or a combination thereof. The wicking member 910 may be treated, soaked, or otherwise saturated with a surfactant prior to being introduced or disposed in the reference conduit cavity 902.

[0317] In some embodiments, grooves or ridges can be constructed on the surface of the wicking member 910, the reference electrode material 808 (e.g., a cured conductive ink), or a combination thereof. Increasing the surface area of ​​the wicking member 910, the reference electrode material 808, or a combination thereof can increase the stability of the half-cell potential of the reference electrode.

[0318] The method may also include supplying or dispensing a conductive ink (Ag-AgCl ink) onto the wick proximal end 914. The conductive ink may be dispensed or otherwise provided to the wick proximal end 914 via a syringe, dropper, dropper dispenser, or pipette. The conductive ink may be dispensed or otherwise provided into a divot, groove, or depression defined by the wick proximal end 914 and a segment of the reference conduit 900 surrounding the wick proximal end 914.

[0319] Dispensing the conductive ink may further include dispensing between about 50 μL and about 500 μL of the conductive ink onto the wick proximal end 914. More specifically, dispensing the conductive ink may further include dispensing between about 80 μL and about 120 μL of the conductive ink onto the wick proximal end 914. For example, the method may include dispensing about 100 μL of the conductive ink onto the wick proximal end 914.

[0320] The conductive ink can then be cured until the conductive ink hardens into the reference electrode material 808. In some embodiments, the method involves curing the conductive ink at a temperature above 100° C. More specifically, the method can include curing the conductive ink at a temperature between about 110° C. and 120° C. For example, the method can include curing the conductive ink at 115° C. The conductive ink can be cured for a time period between about 60 minutes and about 180 minutes to cure the conductive ink. For example, the conductive ink can be cured for a time period between about 110 minutes and about 130 minutes. For example, the conductive ink can be cured for 120 minutes. More specifically, the conductive ink can be cured at 115° C. for 120 minutes.

[0321] A method of measuring solution properties of a sample 102 may include filling a sample chamber 908 of a sample container 802 with the sample 102. The sample 102 may include an infectious agent or a microorganism. The method may also include attaching a reference sensor member 804 configured as a container cap 806 to the sample container 802.

[0322] The vessel cap 806 can include a reference conduit 900 that includes a reference conduit cavity 902, a reference conduit first opening 904, and a reference conduit second opening 906. The reference conduit cavity 902 can be partially filled with a wicking member 910 extending through the reference conduit cavity 902 having a wick distal end 912 and a wick proximal end 914.

[0323] At least a portion of the wick member 910 can be in fluid contact with the sample 102 in the sample chamber. At least a portion of the sample 102 can be pumped by the wicking member 910 toward the wick proximal end 914. A reference electrode material 808 (e.g., a cured conductive ink) can be disposed at the wick proximal end 914. The method can further include electrically coupling the reference electrode material 808 to a parameter analyzer 916 (e.g., a high impedance voltmeter) and electrically coupling the parameter analyzer 916 to an active sensor member 816 that includes an active electrode material 918. At least a portion of the active electrode material 918 can extend into the sample chamber 908 and can be in fluid contact with the sample 102 in the sample chamber 908. As the wicking member 910 pumps or wicks the sample 102, it reaches the reference electrode material 808 and the charge in the sample 102 can establish an electrical connection between the reference electrode material 808 and the active electrode material 918. At this point, a circuit is formed that can then be utilized to perform a measurement of the change in potential of the sample 102.

[0324] The method may further include determining a solution property of the sample 102 based on the potential difference measured between the active electrode material 918 and the reference electrode material 808. As described above, the solution property measured can be the pH of the sample 102, the ORP of the sample 102, or a combination thereof.

[0325] The method may also include pumping air into the sample chamber 908 through a vent 936 defined along at least one of a bottom and a side of the sample container 802. A hydrophobic breathable membrane (e.g., a first breathable membrane 938) may cover the vent 936. The air pumped into the sample chamber 908 may aerate the sample 102 and promote growth of infectious pathogens or microorganisms within the sample 102.

[0326] Air pumped into the sample chamber 908 can exit through an additional breathable membrane (e.g., second breathable membrane 942) covering at least a portion of the bottom surface 950 of the container cap 806, and finally through an air gap 948 (see FIG. 9A) defined between the container cap 806 and the sample container 802 (e.g., through an air gap 948 defined between the male threads of the sample container 802 and the female threads of the container cap 806).

[0327] FIG. 10 shows the change in oxidation-reduction potential (ORP) of three aliquots of a sample measured over time using three different sensor setups. All such samples contained Escherichia coli (E. coli or ECo). One such sensor setup included an active sensor element 816 containing a redox-sensitive material (e.g., an ORP active sensor) as the active electrode material 918, and a commercially available, off-the-shelf silver-silver chloride reference electrode. This sensor setup served as a control, and the results obtained from these measurements served as a standard or benchmark for other measurements of E. coli growth. The other two sensor setups included an active sensor element 816 containing the same redox-sensitive material as the active electrode material, but used a container cap 806 as disclosed herein as the reference sensor element 804. In such cases where the container cap 806 served as the reference sensor member 804, the reference electrode material 808 was a hardened, cured deposit of silver-silver chloride (Ag-AgCl) ink. In such sensor setups, the Ag-AgCl ink used was Ag-AgCl 113-09 developed by Creative Materials, Inc. In other sensor setups, the Ag-AgCl ink used was Ag-AgCl 126-49 developed by Creative Materials, Inc.

[0328] As shown by the three E. coli growth curves, the two sensor setups using the container cap 806 as the reference sensor element performed similarly to the sensor setup using a conventional commercial off-the-shelf silver-silver chloride reference electrode. Any changes in signal timing were within acceptable limits based on the active sensor used.

[0329] 11 is a graph showing cell count results (expressed as colony forming units (CFU) / mL) performed on bacterial colony smears of output samples containing various E. coli strains. In some embodiments, the sensor device 800 disclosed herein can be used as part of a system for preparing output samples of predetermined microbial concentrations, e.g., 0.5 McFarland (MCF) to 0.6 MCF (microbial suspensions exhibiting a turbidity equivalent to 0.5 McF to 0.6 McF), typically used as inputs in antibiotic susceptibility testing.

[0330] The dark colored bars represent quality control (QC) samples that exhibited turbidity of 0.5 McF and 0.6 McF as measured by a standard turbidity (optical density) meter. The QC samples were prepared by resuspending colonies from 18-24 hour culture plates in saline to a final concentration of about 0.5 McF to 0.6 McF (a concentration of about 0.5 McF to 0.6 McF is an acceptable input concentration for antibiotic susceptibility testing). The QC samples consisted of a quality control strain of E. coli known as ATCC 25922. Once such QC samples reached turbidity levels of 0.5 McF and 0.6 McF, such samples were then plated and the number of viable cells (counts) were measured in the plated samples (the results of such counts are displayed on the graph in units of CFU / mL).

[0331] The various white bars in FIG. 11 represent cell counts performed on defined concentrations of output samples prepared using the sensor device 800 disclosed herein. The output samples were prepared from original samples containing various E. coli strains derived from positive blood cultures from septic patients. Approximately 1.50×10 8A defined concentration of CFU / mL (representing a turbidity of 0.5 McF) was set as the target value for all such output samples.

[0332] An original sample containing various E. coli strains was introduced into a sample chamber 908 of a sample container 802 as disclosed herein. A reference sensor member 804 (e.g., container cap 806) was clamped to such sample container 802, and then a parameter analyzer 916 measured or monitored changes in the solution properties of the sample over time.

[0333] The system may also include one or more computing devices communicatively coupled to the parameter analyzer 916. The one or more computing devices may look up a particular lookup table (e.g., a species-specific lookup table) associated with a particular infectious pathogen species or an inter-species or universal lookup table and determine a real-time concentration of the infectious pathogen in the sample chamber 908 based on changes in solution properties of the sample in the sample chamber 908.

[0334] In some embodiments, the concentration data can be determined directly from one or more look-up tables based on real-time changes in solution properties monitored or measured by the parameter analyzer 916, one or more computing devices, or a combination thereof. In these embodiments, the real-time changes in solution properties of the sample in the sample chamber 908 can be directly mapped to threshold solution property changes in the look-up tables, which can be directly related to the estimated concentration of the infectious pathogen in the sample chamber 908.

[0335] In another embodiment, additional calculations and data interpolation can be performed by one or more computing devices based on one or more lookup tables and threshold data from real-time solution property changes measured or monitored by sensor apparatus 800. For example, one method of preparing an output sample of a predetermined concentration may include calculating a sample incubation time or sample preparation time corresponding to the amount of time required for the infectious pathogen in sample chamber 908 to reach a predetermined concentration based on a first threshold time, a second threshold time, concentration data from at least one lookup table, and a desired predetermined concentration. This method is further described in U.S. Patent Application Serial No. 16 / 430,266, the contents of which are incorporated herein by reference in their entirety.

[0336] As can be seen in FIG. 11, smear cell counts performed on output samples (of 0.5 McF) generated in part using the sensor device 800 disclosed herein compared favorably to 0.5 McF and 0.6 McF QC samples generated using the "gold standard" of resuspending colonies from 18-24 hour culture plates and determining the concentration of such suspension using optical density measurements. Nearly all output samples prepared using the sensor device 800 and the disclosed method resulted in samples having infectious agent concentrations within the acceptable range of the QC samples. FIG. 11 shows that the sensor device 800 and the disclosed method can be used to effectively generate output samples of defined concentrations (e.g., 0.5 McF bacterial samples).

[0337] FIG. 12 shows the effect of aeration on the growth rate of two samples 102 containing the aerobic bacterium Pseudomonas aeruginosa (PA). The growth rate was measured based on the turbidity of the samples 102 (with turbidity determined based on the McFarland (McF) reference standard to represent the turbidity of bacterial suspensions). The greater the turbidity of the sample solution, the higher the concentration of PAe in the sample 102.

[0338] As shown in FIG. 12, aeration of the sample 102 (shown by the dashed plot) increased the bacterial growth rate in the sample 102 such that the time it took for the sample 102 to reach a turbidity level equivalent to 0.5 McFarland (McF) was significantly decreased compared to the non-aerated or stagnant sample 102 (shown by the solid plot). The sample 102 can be aerated by pumping a gas (e.g., ambient air, oxygen, etc.) into the sample chamber 908 through the vent 936 and breathable membrane 938. In some embodiments, the vent 936 can be defined on the bottom side of the sample container 802. The gas 940 or ambient air can be pumped into the sample chamber 908 at a constant flow rate of about 1 mL / min to about 10 mL / min.

[0339] 13 shows the effect of aeration on preparation time of two 0.5 McFarland bacterial samples using the sensor device 800 disclosed herein. Such 0.5 McF samples can be prepared for further downstream testing, such as as part of one or more antibiotic susceptibility tests.

[0340] As shown in Figure 13, one sample container 802 filled with a sample containing Pseudomonas aeruginosa (PAe) was vented while another sample container 802 filled with a similar sample containing PAe was not vented. A container cap 806 acting as a reference sensor member 804 was then fastened to each sample container 802, and each sensor device 800 was then electrically coupled to a parameter analyzer 916 used to detect changes in the ORP of the sample.

[0341] A computing device or another device coupled to the parameter analyzer 916 then recorded the ORP changes of the samples within each of the sample containers 802 and compared those values ​​to data in one or more look-up tables read from a computing device coupled to the parameter analyzer 916 or to a database accessible by the computing device.

[0342] 13, the concentration of PAe in the aerated sample container 802 reached the 0.5 McFarland nominal concentration in about 160 minutes, whereas the concentration of PAe in the stagnant or non-aerated sample container 802 reached the 0.5 McFarland nominal concentration in about 460 minutes, demonstrating that aerating the sample container 802 significantly increases the bacterial growth rate in the sample chamber 908.

[0343] Each variation or aspect described or depicted herein has separate components and features which are readily distinguishable from or combinable with the features of any of the other variations or embodiments. Modifications may be made to adapt a particular situation, material, composition of matter, process leading to an object, process acts or steps to the objective, spirit or scope of the present invention.

[0344] Methods referred to herein may be carried out in any order of the recited events that is logically possible, in addition to the recited order of events. For example, the flowcharts or process flows depicted in the Figures do not require the particular order shown to achieve desired results. Moreover, additional steps or operations may be provided or steps or operations may be deleted to achieve desired results.

[0345] It will be understood by those skilled in the art that the methods of the present disclosure may be embodied in whole or in part in a non-transitory (non-transitory) computer-readable or accessible medium, including a device that is readable or executable by a processor or processing unit (PU) of a computing device or other type of machine.

[0346] Furthermore, when a range of numerical values ​​is provided, all intervening values ​​between the upper and lower limits of that range are included. As well as any other stated value or any other intervening value within the stated range, any of the features of the described inventive variations may be set forth and claimed independently or in combination with any one or more of the features described herein.

[0347] All current subject matter mentioned herein (e.g., publications, patents, patent applications, and hardware) is incorporated herein by reference in its entirety, except to the extent that the subject matter is in conflict with that of the present invention, in which case those provided herein take precedence. The referenced items are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such material by virtue of prior invention.

[0348] Reference to an item in the singular includes the possibility of a plurality of that item. More specifically, as used herein and in the appended claims, the singular forms "a" or "an," "said," and "the" include plural referents unless the context clearly dictates otherwise. It should also be noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as an antecedent basis for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements or the use of "negative" limitations. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0349] The present disclosure is not limited in scope to the particular forms described, but is intended to encompass alternatives, modifications, and equivalents of the variations or embodiments described herein. Moreover, the scope of the present disclosure encompasses all other variations or embodiments that may become apparent to one of ordinary skill in the art in view of the present disclosure. The scope of the present invention is limited only by the scope of the appended claims.

Claims

1. a reference sensor member, a container cap configured to be removably coupled to a sample container configured to receive a sample; a reference conduit extending from a bottom surface of the container cap, the reference conduit having a reference conduit cavity; a wicking member disposed within a portion of the reference conduit cavity, the wicking member having a wick distal end and a wick proximal end, wherein when the vessel cap is coupled to the sample vessel, at least a portion of the wicking member is in fluid communication with the sample, and at least a substantial amount of the sample is drawn by the wicking member toward the wick proximal end; a reference electrode material disposed at the wick proximal end, the reference electrode material configured to exhibit a substantially stable electrode potential relative to an active electrode electrically coupled to the reference electrode material; and A reference sensor member comprising:

2. 2. The reference sensor element of claim 1, wherein said container cap is made in part of a non-conductive material.

3. 3. The reference sensor element of claim 1 or 2, wherein the reservoir cap is made in part of a transparent, non-conductive material such that at least a portion of the wicking element is visible through the reservoir cap.

4. 10. The reference sensor element of claim 1, wherein the reference electrode material is a conductive ink provided or dispensed on the wick proximal end.

5. The reference sensor element of claim 4 , wherein the conductive ink provided or dispensed on the wick proximal end is hardened by curing.

6. 5. The reference sensor element of claim 4, wherein said conductive ink is a silver-silver chloride ink.

7. 10. The reference sensor element of claim 1, wherein said wicking element is made in part from a porous polymeric material.

8. 10. The reference sensor element of claim 1, wherein said wicking element is made in part of natural fibers.

9. 10. The reference sensor element of claim 1, wherein said wicking element comprises pores sized from about 15 μm to about 150 μm.

10. 2. The reference sensor element of claim 1, wherein the wicking element is treated with a surfactant such that at least a surface of the wicking element is covered with the surfactant, wherein the surfactant is configured to increase the hydrophilicity of the wicking element.

11. 1. A method for measuring solution properties of a sample, comprising: filling a sample chamber of a sample container with a sample containing an infectious pathogen; attaching a container cap to the sample container; wherein the vessel cap includes a reference conduit having a reference conduit cavity, a reference conduit first opening, and a reference conduit second opening; wherein the reference conduit cavity is partially filled with a wicking member having a wick distal end and a wick proximal end and extending through the reference conduit cavity; wherein at least a portion of the wicking member is in fluid contact with the sample in the sample chamber, and at least a substantial amount of the sample is drawn by the wicking member toward the wick proximal end; wherein a reference electrode material is disposed at the proximal end of the wick; electrically coupling the reference electrode material to a parameter analyzer, and electrically coupling the parameter analyzer to an active sensor element including an active electrode material; wherein at least a portion of the active electrode material extends into the sample chamber and is in fluid contact with the sample; determining a solution property of the sample based on the potential difference measured between the active electrode material and the reference electrode material; The method includes:

12. The method of claim 11 , wherein the reference electrode material is a conductive ink provided or dispensed on the wick proximal end.

13. The method of claim 12 , wherein the conductive ink provided or dispensed onto the wick proximal end is hardened by curing.

14. The method of claim 12, wherein the conductive ink is a silver-silver chloride ink.

15. 12. The method of claim 11, wherein air is pumped into the sample chamber through a vent hole defined along at least one of the bottom and side sides of the sample container and through a hydrophobic, breathable membrane covering the vent hole, wherein the air pumped into the sample chamber aers the sample.

16. 16. The method of claim 15, wherein air pumped into the sample chamber is exhausted from the sample chamber through an additional breathable membrane covering at least a portion of the bottom surface of the container cap and through a gap defined between the container cap and the sample container along the attachment connection.

17. 12. The method of claim 11, wherein determining the solution properties of the sample comprises measuring the pH of the sample, and the active electrode material is made in part of a pH-sensitive material.

18. 18. The method of claim 17, wherein the pH-sensitive material comprises at least one of silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium dioxide, ruthenium dioxide, and zirconium dioxide.

19. 12. The method of claim 11, wherein determining the solution properties of the sample comprises measuring the oxidation-reduction potential (ORP) of the sample, and wherein the active electrode material is made in part of a redox-sensitive material.

20. 20. The method of claim 19, wherein the redox-sensitive material comprises at least one of platinum, gold, silicon dioxide, aluminum oxide, titanium dioxide, tantalum pentoxide, hafnium dioxide, iridium dioxide, ruthenium dioxide, and zirconium dioxide.

Images