Patent Application
20230408432
The present invention is within the field of real-time detection of growth of microorganisms such as bacteria or biofilms in containers with insulated walls of commercial labware such as laboratory flasks, microwell plates and laboratory vials, dishes etc. More specifically, embodiments of the present invention are a docking system adapted to dock containers with electrically insulated walls and comprising microorganisms reproducibly into a docking station of a docking system. Positioning electrodes of the docking system at preselected positions on the insulated walls of the containers enables reproducible real-time analyses of growth of microorganisms by an impedance analyser connected to the device.
When growing bacteria in biotechnology applications, microorganisms and their by-products need to be sampled and analysed [C. E. Turick et al. Appl. Microbiol. Biotechnol. 103, 8327-8338 (2019)]. Commonly used methods include biochemical phenotype methods, molecular hybridization methods and amplification methods [M. Simić et al. IEEE Sensors J. 20, 12791-12797 (2020)], but they are time consuming, labour intensive and costly.
Bacteria have a wide variety of shapes, dimensions and compositions [R. Gnaim et al. BioTechniques 69, 27-36 (2020)]. Various techniques have been developed for faster and less labour-intensive determination of bacterial concentration, including fluorescence cytometry (WO 95/31481) and image-analysis (U.S. Pat. No. 8,780,181). Other methods are cell counting, Coulter counters, pH monitoring, magnetic, electrochemical and bioluminescence [R. Narang et al. Sci. Rep. 8, 15807 (2018)]. These techniques require extensive image or signal processing as well as large and expensive instrumentation, which is laborious to work with and lack the potential for miniaturization. In addition, special effort is required to manufacture custom systems in which electrodes are exposed to growing bacteria.
Non-labelling detection methods are simpler to work with and include quartz crystal microbalance and surface plasmon resonance techniques. However, those methods also require that the bacterial growth platforms are adapted to these instruments.
During bacterial growth, the structural and functional changes that occur to a cell during growth can be detected by electrochemical analysers, which enable the monitoring of the bioprocesses in real-time. Applying electrical signals to living cells causes reactions with the components of the cells and frequency dependent polarization, which can be related to the polar nature of lipid membranes, proton gradients on the outer surface of viable cells, and membrane-associated electron transfer reactions linked to metabolic activity. Simplified, this behaviour of the cells can be described in terms of capacitance, electrical permittivity and conductivity. The capacitance to resistance ratio changes during cellular growth and reflects factors related to biomass density, cell viability, membrane integrity and the overall metabolic state of microbes. Dead cells or non-biomass solids that do not pose an intact plasma membrane do not polarize and therefore do not have a significant capacitance relative to the cell suspension [J. Song et al. ACS Sens. 5, 1795-1803 (2020)].
The variation in capacitance and resistance can readily be measured by an analyser using electrochemical-impedance-spectroscopy (EIS). EIS is a fast, sensitive, and label-free technique that can characterize (bio)physical processes at the substrate-biomaterial interface. For standard impedance spectra, at low frequencies (up to several tens of kHz), the effects of a double layer (metal-medium interface effects) are dominant. At medium frequencies (several tens of kHz to several MHz), the impedance depends on the medium and cell/particle. There is a first plateau, which depends on cell size, and a second plateau, which depends on cell cytoplasm properties. At frequencies above several MHz, the parasitic effects due to the substrate and electrode connections are dominant [A. L. A. de Araujo et al. Biosensors 9, 108, 1-4 (2019)]. However, for analysers based on EIS, microbial growth on surfaces has been demonstrated to be sensitive to the choice of metal electrode material such as stainless steel, indium-tin-oxide, platinum and gold [J. Song et al. ACS Sens. 5, 1795-1803 (2020)].
An online monitoring of bacterial growth based on a multichannel capacitively coupled contactless conductivity detector and eight bacterial culture tubes, where the electrodes are not in direct contact with the measured solutions has been reported [Zhang et al., Anal. Chem. 2018, 90, 10, 6006-6011]. The detector for each tube is based on a couple of copper cylinders with internal diameter of 5.01, thickness of 0.4 mm and a length of mm and with a spacing of 46.0 mm. GB 2 260 407 describes non-contacting, bridgeless methods which utilize concentric multi-electrode or multi-coil structures for capacitive and inductive measurement on samples of matter without contact together with the use of multiple-frequency excitations to separate out or reduce effects of ionic conductivity and/or multiple dielectric dispersions in samples. From any such dispersion or frequency region of interest, the methods yield physical or chemical properties of the sample, e.g. biomass in water. However, the method described in GB 2 260 407 is not suitable for detecting growth of microorganisms in off-the-shelf labware.
US 2006/0216203 describes a multi-well impedance measurement device for real-time detection of cellular activation with a plurality of wells and/or chambers and with a plurality of electrodes lying on the bottom surface of each plurality of wells/chambers. However, the electrodes are exposed to the samples as measured by the device. The system is based on contact impedance measuring electrodes, where the electrodes are in physical contact with the samples, and they cannot be attached to and detached from the wells comprising the cells growing at the bottom of the wells.
US 2009/0149334 describes a method of measuring properties of a liquid contained in individual wells inside a multi-well array, where capacitor electrodes are provided in the multi-well array. The method is based on measuring capacitance inside each individual well, where the electrodes are adapted to detect a capacitance value of each individual well without interference of neighbouring wells. The property can be a volume of the liquid inside the individual wells, a state of presence of the liquid inside the individual wells or further a change of volume of the liquid inside the individual wells. However, this invention is not applicable to wells with insulated walls in off-the-shelf labware.
EP 3 301 438 describes a sensor and method for detecting a state of a fluid within a microfluidic plate, comprising at least a first planar electrode and a second planar electrode arranged parallel to one another to form an electric capacitor and configured to generate an electric field within the fluid. The sensor is based on detecting the state of the fluid by means of a capacitance of the electric capacitor.
The state of the fluid comprises at least one element of the group consisting of presence of the fluid, absence of the fluid, predetermined mixture of the fluid with at least one component different from the fluid, presence of an enclosure of the fluid, absence of an enclosure of the fluid, fill level of the fluid, and flow rate of the fluid. The fluid may be a liquid, particularly a biological liquid. The sensor comprises planar electrodes, which do not contact the fluid and may be arranged parallel to the microfluidic plate.
However, EP 3 301 438 does not teach how to attach and detach the connection between the wells comprising the fluid and the planar electrodes and how the electrodes are positioned with respect to the microfluidic plate comprising the fluid, which is required for a reliable detection of growth of microorganisms. In addition, the invention is not applicable to off-the-shelf labware.
U.S. Pat. No. 9,400,272 describes a non-invasive method of detecting microbial growth by monitoring changes in the dielectric constant of the specimen caused by metabolic processes. Changes in the dielectric constant of a biological sample in a growth medium or minute changes to a biological sample's dielectric constant are measured e.g. by varying the electric field capable of sensing a change in the dielectric constant between capacitive electrodes.
In one embodiment, the interdigitated electrodes are provided on a flexible circuit substrate such as mylar or Kevlar film, and the electrodes are wrapped around the vessel or bottle such that an electrode surface area is in physical proximity to the liquid sample inside, where the dielectric constant of the sample changes with microorganism growth and metabolite release. In another embodiment, plates include opposing pairs of conductive electrodes, wherein each pair of electrodes are placed above and below microwells. However, U.S. Pat. No. 9,400,272 does not teach how to reproducibly attach and detach the vials, vessels, bottles or microwells with respect to the electrodes and how to minimize and/or reproduce the spacing between the electrodes and the surfaces of the vials, vessels, bottles of microwells by establishing contact interfaces. Since any variations in the electrode positions and spacing will cause a change in the capacitance, U.S. Pat. No. 9,400,272 is not adapted to make quantitative measurements of microorganisms. In addition, the invention is not applicable to off-the-shelf labware.
US patent application 2019/0359970 describes an invention of a cuvette comprising a body of a nonconductive material, a sample cavity configured to receive a sample, a first wall configured to couple to a first electrode of a pulse generating system that comprises a capacitive element, and a second wall configured to couple to a second electrode of a pulse generating system. In some embodiments of the invention, the cuvette is a container configured to hold a sample which may be disposed between two electrodes, which are spaced apart on opposite sides of the container, and a sample holder may a spring-loaded mechanism that pushes electrodes of the sample holder against the two electrodes. However, in these embodiments, the cuvette comprises electrodes in physical contact with the sample to be measured, and the invention is not applicable to off-the-shelf labware.
There is a need of rapid, real-time, high-throughput and non-contact detection of microbial growth, which can be used with off-the-shelf labware with insulated walls, including laboratory bottles, flasks, T-flasks, vials, dishes and plates. There is therefore a need of a device enabling the attachment and detachment of containers comprising microorganism as integrated with off-the-shelf labware to an analyser for real-time detection of microbial growth reproducibly. There is also a need of ensuring reproducibly positioning and with minimized spacing between the electrodes and the insulated walls of the containers and with no physical contact between the electrodes and the microorganisms. It has been found that this objective may be achieved by means of locating at least one of the electrodes at the bottom side of a container, thereby making it readily to attach and detach the electrodes to and from the container reproducibly with minimum spacing between the electrodes and the outer surface of the container. This enables reproducibly quantitative determination of growth of microorganisms.
These and other object needs have been fulfilled by the invention or embodiments thereof as defined in the claims and/or as described herein below.
It has been found that the invention or embodiments thereof have a number of additional advantages, which will be clear to the skilled person from the following description.
In an embodiment of the present invention of a docking system for electrochemical detection of growth of microorganisms in a container, a docking system comprises a docking station, a set of electrodes comprising at least two electrodes and a docking arrangement. The docking arrangement comprises at least one movable element and a set of electrical connectors adapted for connecting the respective electrodes to analyser, and the docking arrangement is adapted for temporarily holding a container in location relative to the set of electrodes, to provide the respective electrode to be in physical contact with an outer surface of said container at respective preselected locations, wherein the electrodes are physically separated from each other. The docking arrangement is connected to or is connectable to the docking station, and the docking station may comprise a docking site for a container, wherein the docking site may comprise a bottom structure comprising at least one electrode of the set of electrodes.
In another embodiment of the present invention of a docking system, one or more electrodes of the set of electrodes are stationary in the docking station at a docking site to ensure that the electrode comes into physical contact with the outer surface of a container when located at the docking site in the docking station, and the stationary electrode preferably forms part of a bottom structure of the docking site.
The docking arrangement in the docking system may be correlated to or engaged with at least one of the electrodes of the set of electrodes, to provide the physical contact between the container outer surface and the electrode, and the at least one movable element of the docking arrangement has a first position where at least one of the electrodes of the set of electrodes is detached from the outer surface of the container and a second position where the at least one electrode of the set of electrodes is held in physical contact with the outer surface of the container at an essentially constant pressure. The pressure between the set of electrodes and the outer surface of the container is maintained for a selected period of time.
In further embodiments of the present invention, the set of electrodes may be substantially plane and is preferably adapted to be in physical contact with a plane portion of the outer surface of a container, such as a bottom portion of the outer surface of a container. One of the electrodes of the set of electrodes may be circular, disc or ring shaped and one electrode of the set of electrodes may comprise a strip shaped electrode adapted for encircling and contacting an outer surface of the container, wherein the strip shaped electrode is associated to the docking arrangement, such that the strip shaped electrode is detached from the outer surface of the container when the docking arrangement is in a first position and is contacting the outer surface of the container when the docking arrangement is in a second position.
The docking arrangement in even further embodiments of the present invention comprises a spring element adapted, wherein said spring element is adapted to establish a pressure between at least one of the electrodes of the set of electrodes and an outer surface of the container, such as an outer surface of a bottom area of the container. The docking arrangement may comprise at least one constraining element adapted for holding the container in temporarily fixed condition, wherein the constraining element(s) preferably is adapted for holding the container in the temporarily fixed condition while the at least one movable element of the docking arrangement to establish a pressure between at least one of the electrodes of the set of electrodes and an outer surface of the container. The docking system may be adapted for electrochemical detection of growth of microorganisms in two or more containers, such as in two or more sub-containers of a multi container device, wherein the docking arrangement is adapted for temporarily holding said containers in location relative to respective sets of electrodes, to provide the respective electrode of the sets of electrodes to be in physical contact with an outer surface of said respective containers at respective preselected locations, wherein the electrodes of respective sets of electrodes are physically separated from each other.
In even further embodiments of the present invention, the system comprises a foil with a pressure sensitive adhesive adapted for being attached the outer surface of the container, preferably at a bottom location of the container and wherein an electrode of the set of electrodes are fixed on said foil.
In another aspect of the present invention, a method is provided for electrochemical detection of growth of microorganisms in a sample in a container. The method comprises docking the container in a docking system, providing the docking arrangement for temporarily holding the container in location relative to the set of electrodes, to provide the respective electrode to be in physical contact with an outer surface of said container at respective preselected locations, wherein the electrodes are physically separated from each other, connecting the respective electrical connectors to an analyser; performing at least one electrical measurement and performing the detection of growth of microorganisms based on said at least one measurement.
Throughout the description or claims, the singular encompasses the plural and the plural encompasses the singular unless otherwise specified or required by the context.
The container may be, but is not limited to a bottle, a laboratory flask, a well of a microwell plate, a vial or the like. It may also be a tube or any hollow item, which contains a fluid or can be filled or partly filled with a fluid including a spacing between two plane plates. It may further be the top of a plane plate such as a microscope slide, where the surface tension of the liquid enables the liquid to be placed on the top surface of the plane plate; or it may be a concave plate or any item, which has the ability of containing a liquid. The container may be transparent, opaque or non-transparent for light. The present invention also covers arrays of containers, which may have essentially the same sizes and shapes, and arrays of containers with different sizes and shapes. The containers referred to in the description of the present invention comprise at least one electrically insulated wall through which the detection may be made. The insulated wall may be of a material, which is not electrically conductive, including but not limited to a glass, a polymer, an adhesive, or any combination comprising one or more of those materials.
In the following, “electrical connection” between two electrical elements, such as between an electrode of electrically conductive materials and an electrical connector should be understood as an ohmic or galvanic contact that allows electrical current to flow between two electrical elements, and with a reproducible and ideally negligible contact resistance and voltage drop. The tern “negligible” means that the contact resistance and associated voltage drop can be compensated for by an analyser, and that the contribution to the measurement uncertainty from the variations of the contact resistance and associated voltage drop is within acceptable limits. Acceptable limits mean within 10%, more preferably within 1% and even more preferably within 0.1%. The actual value of how much contact resistance and associated voltage drop can be accepted depends on the impedance level, among others, and on the terminal configuration between the analyser used and the electrical connector. The “electrical connection” may be made by establishing a low electrical contact interface and/or a resistance between the two elements by soldering, bonding including ionic, covalent, and metallic bonding, gluing using electrically conductive glues or adhesives including but not limited to silver, gold, aluminium or the like; using electrical connectors with mechanical attachment, or by applying a sufficient mechanical force between surfaces of the two electrical elements, e.g. by means of a spring or the like. Other wordings used, which should be understood in the same sense, are “electrically connected” or simply “connected”. The wording “A is connectable to B”, where A and B are two items, means that a connection can be established between item A and item B.
In the following, the term “microorganisms” should be interpreted to comprise organisms such as bacteria, protozoa, algae, fungi, cells, and yeast or the like, covering but not exclusively organisms, which can be observed through an optical microscope or an electron microscope. The present invention covers but not exclusively electrochemical detection of growth of such microorganisms in a container.
At the position where the first electrode 3 is detached from the outer surface of the bottom of the bottle 4, and the second electrode 5 is detached from the outer surface of the cylindrical wall of the bottle 7, the docking arrangement is in the first position. At the position where the first electrode 3 is attached to a preselected location on the outer surface of the bottom of the bottle 4, and the second electrode is attached around a preselected location on the outer surface of a part of the cylindrical wall of the bottle 7, the docking arrangement is in the second position.
In the embodiment as illustrated in
Embodiments of the present invention include docking systems comprising docking stations and docking arrangements for attaching and detaching containers such as bottles or other types of labware to one or more sets of electrodes establishing the electrical connection. Such docking arrangements may consist of, but are not limited to spring loaded levers, pads, blades, and rollers, which exert pressure on the contact interfaces and ensure minimized air spacing between the sets of electrodes and the insulated walls of the containers. Embodiments of the present invention also include docking systems comprising docking arrangements for attaching and detaching containers controlled manually or by electrical or pneumatic actuators, pneumatic suction through pads, cups or the like, grommets of suitable shape and size, and electromagnets that keep ferrous materials attached to or being part of the containers. Embodiments of the present invention further include docking arrangements comprising clips or clamps, bayonet or threaded mounts, or screws with or without additional tension provided by means such as pneumatic, electrical actuators or operated manually; machined parts providing press-fit, or having slits, slots or other physical structures that allow containers to be positioned in the docking station with preselected positions of the electrodes on the surface of the outer wall of the container. In addition, the docking device may include elements to detect or adjust the positioning of the containers, or elements to check the electrical connections.
The bottle 2 can be detached from the docking station 1 by loosening the docking screw and the diameter 9 of the ring element 6 becomes larger than the outer diameter of the cylindrical wall 11 of the bottle 2. The bottle 2 can be attached to the docking station 1 by tightening the docking screw 10, and the diameter 9 of the ring element 6 is reduced with an inner diameter 12 essentially equal to the outer diameter of the bottle 11. This may be made used a mechanical tool combined with or without a motorized drive. It may also be combined with a torque wrench to ensure that the tightening has the right torque and that the torque is reproduced between various mounting of bottles.
An excessive torque may produce deformation or cracks in the wall of the bottle. A right torque means producing a suitable tightening pressure at the contact interface between the strip shaped second electrode 5 and the part of cylindrical outer wall of the glass to which the second electrode 5 is attached, and the air spacing between the second electrode 5 and the walls of the bottle 7 is minimized.
Gravity enables tight attachment of the circular disc shaped first electrode 3 to the preselected location at the surface of the outer wall bottom of the bottle 4. Alternatively, the docking screw 10 may be further adapted to enable a suitable tightening pressure at the docking site 20 onto the preselected location at the surface of the outer wall of the bottle to the first electrode 3. The first circular disc shaped electrode 3 may further comprise a planar surface or a curved surface adapted to minimize air spacing to the surface of the outer wall of the bottom of the bottle 4 or the circular disc shaped first electrode 3 may further comprise a combination of a thin conductive film on top of a flexible material such as a polymer foam combined with an adhesive such as a pressure sensitive adhesive. The circular disc shaped first electrode 3 may further comprise a spring element adapted for applying a mechanical force between the top surface of the first electrode 3 and the surface of the outer wall of the bottom of the bottle 4.
A guard ring of electrically conductive material 13 surrounds the first electrode 3 and minimizes stray electric fields. This is the preferred positioning of a guard ring. Alternatively, a guard may be positioned with parts above and below the strip shaped second electrode 5. An additional guard may be disposed around the docking station including covering the bottle. Design of guards with the purpose of reducing electrically stray fields is commonly used for capacitance sensors and it is well-known for a person skilled in the art.
The first electrode 3 is electrically connected or connectable to a first electrical connector 14 preferably made by a coaxial cable based on conductive material such as copper with the shield connected or connectable to the guard of the analyser used and the inner wire connected or connectable to the electrode 3 and with a suitable termination such as, but not limited to a BNC, SMA or SMB connector. The second electrode 5 is electrically connected or connectable to a second electrical connector 15 preferably made by a coaxial cable with the shield connected or connectable to the guard of the analyser used and the inner wire connected or connectable to the second electrical connector 15 and with a suitable termination such as, but not limited to a BNC, SMA or SMB connector. The first electrical connector 14 and the second electrical connector 15 are one set of electrical connectors. The guard ring 13 is electrically connected or connectable to a third electrical connector 16 with a suitable termination such as, but not limited to a BNC, SMA or SMB connector, which is preferably connected or connectable to the common earth or to the guard of the analyser used.
The set of electrodes 3 and 5 and the guard 13 may be made of a metal with high conductivity such as, but not limited to stainless steel, copper, aluminium, gold, silver or the like, or carbon-based conductive materials such as, but not limited to graphite, graphene, reduced graphene oxide-carbon or the like. Alternatively, the set of electrodes 3 and 5 and the guard 13 may be of conductive polymer materials such as, but not limited to polyacetylene, polyaniline, polythiophene, polypyrrole, polyfuran, poly(para-phenylene), and poly(phenylenevinylene).
For attachment of the second strip shaped electrode 5 around a preselected location on the surface of a part of the cylindrical outer wall of the bottle 7, a suitable tightening pressure is preferably in the range 10 N/m 2-300 000 N/m2, more preferably in the range 100 N/m 2-30 000 N/m2, and even more preferably in the range 1 000 N/m 2-10 000 N/m2. As it is well-known for a person skilled in the art, depending on the diameter of the ring element 12 and the height of the ring element 17, the suitable pressure can be converted to a suitable torque on the docking screw 10. The height of the second electrode 17 should preferably be in the range from 1/10000-⅓ of the height of the bottle 18, more preferably be in the range from 3/1000-⅛ of the height of the bottle 18, and even more preferably in the range from 1/100- 5/100 of the height of the bottle 18.
When docking a bottle 2 into the docking station 1 with the docking arrangement being in the second position, a preselected and reproducible tightening pressure is maintained temporarily by gravity and the ring element 6 between the disc shaped first electrode 3 and the outer surface of the bottom of the bottle 4, and between the strip shaped second electrode 5 and part of the cylindrical outer wall of the bottle 7, for a time period of at least 1 minute, such as at least 10 minutes. The time period should preferably be of the same order as or exceed the lag phase, the experimental growth phase and the stationary phase of bacterial growth.
There may be variations in the sizes and diameters of the bottles used with the present embodiment of the invention. The diameters of the docking station and the docking arrangement should advantageously be adapted to accept the variations in the diameters of the bottles used. The variation in the movable element 8 should advantageously be sufficiently wide to cover the tolerances or accept the variations in the outer diameters of the bottles used.
From the diameter of the first electrode 3, the height 17 and the diameter 9 of the second electrode 5 and the distance 19 between the first electrode 3 and the second electrode 5, a cell constant can be computed. Alternatively, the cell constant can be determined by measuring the impedance between the first electrical connector 14 and the second electrical connector 15 with the bottle 2 filled with a solution of a reference fluid of known electrolytic conductivity. As it is known by a person skilled in the art, the cell constant as determined between two electrodes multiplied by the capacitance as determined between the same two electrodes is equal to the effective dielectric constant, and determination of cell constant is equivalent to determination of capacitance.
The geometrical dimensions of the first electrode 3, the second electrode 5, the guard 13 and the distance between the first electrode and the second electrode 19 defining the cell constant should be adapted for real time detection of growth of microorganisms according to the electrolytic conductivity of the fluid inside the bottle 2 comprising the microorganisms to be detected. In “C. Thirstrup and L. Deleebeeck, IEEE Trans. Instrum. Meas. 70, 1008222 (2021)”, it has been reviewed how to compute cell constants and how to determine cell constants using reference fluids. The contents and references therein are incorporated in the present invention.
At the position where the first electrode 22 is detached from the outer surface of the bottom of the bottle 4′, the second electrode 23 is detached from the outer surface of the cylindrical wall of the bottle 7′, the third electrode 27 is detached from the outer surface of the cylindrical wall of the bottle 7′, the docking arrangement is in a first position. At the position where the first electrode 22 is attached to a preselected location on the outer surface of the bottom of the bottle 4, the second electrode 23 is attached around a preselected location on the outer surface of a part of the cylindrical wall of the bottle 7′, and the third electrode 27 is attached around a preselected location on the outer surface of a part of the cylindrical wall of the bottle 7′, the docking arrangement is in a second position.
In the embodiment as illustrated in
The first docking screw 31 enables loosening and tightening the first ring element 24 around the first part of the cylindrical outer wall 7′ of the bottle 2′ and ensures tight attachment of the strip shaped second electrode 23 to the first part of the cylindrical outer wall of the bottle 7′. The second docking screw 32 enables loosening and tightening the second ring element 28 around the second part of the cylindrical outer wall of the bottle 7′ and ensures tight attachment of the second strip shaped electrode 27 to the second part of the cylindrical outer wall of the bottle 7′.
The bottle 2′ may be detached from the docking station 21 by loosening the first docking screw 31, and the diameter 26 of the first ring element 24 becomes larger than the diameter of the cylindrical outer wall 11′ of the bottle 2′; and by loosening the second docking screw 32, and the diameter 30 of the second ring element 28 becomes larger than the diameter of the cylindrical outer wall 11′ of the bottle 2′. The bottle 2′ can be attached to the docking station 21, by tightening the first docking screw 31, and the diameter 26 of the first ring element 24 is reduced to an inner diameter essentially equal to the outer diameter of the bottle 11′; and by tightening the second docking screw 32, and the diameter 30 of the second ring element 28 is reduced to an inner diameter essentially equal to the outer diameter of the bottle 11′. This may be made using a mechanical tool without or combined with a motorized drive. It may also be combined with the use of a torque wrench to ensure that the tightening of the first ring element 24 and the second ring element 28 have the right torques, and that the torques can be reproduced between various mounting of bottles. The phrase “right torques” mean herein producing a suitable tightening pressure at the contact interfaces between the strip shaped second electrode 23 and the first part of cylindrical outer wall of the bottle to which the second electrode 23 is attached, and between the strip shaped third electrode 27 and the second part of cylindrical outer wall of the bottle to which the third electrode 27 is attached; thereby providing that the air spacing between the second electrode 23 and the outer wall of the bottle 7 respectively between the third electrode 27 and the outer wall of the bottle 7 are minimized.
Gravity enables tight attachment of the circular disc shaped first electrode 22 to the preselected location at the surface of the outer wall of the bottom of the bottle 4′. Alternatively, the first docking screw 31 and/or the second docking screw 32 may be adapted to enable a suitable tightening pressure at the docking site 40 between the preselected location at the outer surface of the bottle and the circular disc shaped first electrode 22. The circular disc shaped first electrode 22 may further comprise a planar surface or a curved surface adapted to minimize air spacing to the surface of the outer wall of the bottom of the bottle 4′, or the circular shaped first electrode 22 may further comprise a combination of a thin conductive film on top of a flexible material, such as a polymer foam combined with an adhesive such as a pressure sensitive adhesive. The circular disc shaped first electrode 22 may further comprise a spring element adapted for applying a mechanical force between the top surface of the first electrode 22 and the surface of the outer wall of the bottom of the bottle 4′.
A guard ring of electrically conductive materials 33 surrounds the first electrode 22 and minimizes stray electric fields. This is the preferred location of a guard ring.
Alternatively, a first guard may comprise parts positioned above and below the second electrode 24, and a second guard may comprise parts positioned above and below the third electrode 27. An additional guard may be disposed around the docking station including covering the bottle 2′.
The embodiment as illustrated schematically in
The sets of electrodes 22, 23, 27 and the guard 33 may be made of a metal with high electrical conductivity such as, but not limited to stainless steel, copper, aluminium, gold, silver or the like, or carbon-based conductive materials such as, but not limited to graphite, graphene, reduced graphene oxide-carbon or the like. Alternatively, the sets of electrodes 22, 23, 27 and the guard 33 may be of conductive polymer materials such as but not limited to polyacetylene, polyaniline, polythiophene, polypyrrole, polyfuran, poly(para-phenylene), and poly(phenylenevinylene).
For attachment of the second electrode 23 around a first part of the cylindrical outer wall of a bottle 7′ and attachment of the third electrode 27 around a second part of the cylindrical outer wall of the bottle 7′, a suitable tightening pressure is preferably in the range 10 N/m 2-300 000 N/m2, more preferably in the range 100 N/m 2-30 000 N/m2, and even more preferably in the range 1 000 N/m 2-10 000 N/m2. The height of the strip shaped second electrode 38 and the height of the strip shaped third electrode 39 should preferably be in the range from 1/10000-⅓ of the height of the bottle 18′, more preferably be in the range from 3/1000-⅛ of the height of the bottle 18′, and even more preferably in the range from 1/100- 5/100 of the height of the bottle 18′. The height 38 of the second electrode 24 and the height 39 of the third electrode 28 may be essentially identical or they may be different.
The cell constant as measured between the first electrical connector 34 and the second electrical connector 35 is smaller than the cell constant as measured between the first electrical connector 34 and the third electrical connector 36. A cell constant can also be measured from the second electrical connector 35 and the third electrical connector 36. The embodiment of the present invention as illustrated schematically in
Other embodiments of the present invention may include three, four or an even larger multitude of electrodes and a docking arrangement comprising a multitude of movable ring elements, each ring element being adapted to be attached around a preselected location on the outer surface of a part the cylindrical outer wall of the bottle 7′, and to be detached from the preselected location on the outer surface of the part the cylindrical outer wall of the bottle 7′. The docking arrangement enables increasing and decreasing the diameter of each ring element in order for the multitude of electrodes to be attached to and detached from the bottle 2′.
The bottle 2 and 2′ may be a standard bottle, e.g. from Schott Duran made of borosilicate glass, with volumes in the range from 5 mL-10 L, where the wall thickness of the bottle typically is in the range from 1 mm to 3 mm, or the bottles 2 and 2′ may be vials made of borosilicate glass or quartz with volumes in the range from 2 mL to 20 mL, where the wall thickness of the vial typically is 1 mm. The bottle material may also be other types of glass such as, but not limited to silicon dioxide, silicates, soda lime or aluminosilicate, or polymers such as, but not limited to low-density-polyethylene, high-density-polyethylene, polypropylene, polycarbonate, polystyrene and acrylics.
The present invention also includes embodiments, where a magnet is immersed in the bottle 2 or 2′ or any other container, which combined with a magnetic stirrer ensures stirring of the fluid inside the container, and the first electrode 3 or 22 should preferably be made of a non-magnetically conductive material including, but not limited to aluminium, gold, silver, copper, titanium and zinc, non-magnetic stainless steel such as SUS305, alloys such as brass and bronze; conductive polymers and non-magnetic carbon compounds. The present invention also includes embodiments, where the fluid inside the container is mixed by an orbital shaker, an acoustic mixer or other types of mixers.
In the embodiment of the invention as illustrated in
At the position where the first rail 55 is detached from the first side of the microwell plate 58, the second rail 56 is detached from the second side of the microwell plate 59, and the third rail 57 is detached from the third side of the microwell plate 60, the docking arrangement is in a first position. At the position where the first rail 55 is attached to the first side of the microwell plate 58, the second rail 56 is attached to the second side of the microwell plate 59, and the third rail 57 is attached to the third side of the microwell plate 60, the docking arrangement is in a second position.
In some embodiments of the present invention, the first rail 55 and the second rail 56 in combination with a constraining element 61 ensure sufficient force on the first side of a microwell plate 58 and the second side of a microwell plate 59 to reproducibly hold the microwell plate positioned with a sufficient pressure against each set of first electrode 44 and second electrode 45. The constraining element 61 may act as a spring and is preferably made from an elastic material such as a polymer or a metal such as stainless steel.
Other embodiments may comprise one or more elastic elements or spring elements being adapted on one or more rails ensuring reproducibly holding the microwell plate positioned with a sufficient pressure against each set of first electrode 44 and second electrode 45. When the microwell plate is docked, the bottom surfaces of each container 52 should preferably be in physical contact with the plane surface of the docking station 62. This can be provided by matching the height position of each first movable pin body 46 and each second movable pin body 47 with the plane surface of the docking station 62.
Each set of first movable pin element 46′ and second movable pin element 47′ may be mounted on a printed-circuit-board comprising a suitable layout of a multitude of electrical connectors ensuring electrical connection to each set of electrodes 44 and 45. The movable pin elements may be, but are not limited to two-part spring probes with a length in the range from 1 mm-200 mm, a diameter in the range from ø0.2 mm-ø5.0 mm and a head with a diameter in the range from ø0.05 mm-20 mm, and a spring force in the range from 0.1 N-100 N. The material may be, but is not limited to, a metal such as gold, silver, aluminium, or an alloy such as, but not limited to phosphor bronze. The selection includes an RS-PRO Rounded 2-part Spring probe with a length of 25 mm, a diameter of ø1.36 mm, a head of ø1.8 mm, a spring force of 1.47 N, and a contact resistance of 30 mΩ.
The disc shaped first electrode 66 is embedded at least partly inside the cylindrical shaped second electrode 67, and the inner diameter of the circular conductive cylinder 74 is larger than the outer diameter of the circular conductive rod 71. At least two of the electrical connectors must be physically separated and with essentially no direct electrical connection to each other. Part of the guard may be in between, but without physical contact to the first electrode 66 and the second electrode 67.
The disc shaped first electrode 68 is embedded at least partly inside the cylindrical shaped second electrode 69, and the second electrode 69 is embedded at least partly inside the cylindrical shaped third electrode 70. The inner diameter of the first circular conductive cylinder 82 is larger than the outer diameter of the circular conductive rod 79 and the inner diameter of the second circular conductive cylinder 85 is larger than outer diameter of the first circular conductive cylinder 82. At least two of the electrical connectors must be physically separated and with essentially no direct electrical connection to each other. Part of the ground plane may be in between but without direct electrical connection to the first electrode 68, the second electrode 69 and the third electrode 70.
The present invention also includes embodiments with multitudes of circular conductive cylinders with increasing inner diameters, which are larger than the outer diameter of the circular conductive cylinders being embedded at least partly inside. The present invention also includes embodiments, where the circular conductive rod 71,79 is replaced by a circular conductive cylinder or other configurations such as, but not limited to a needle with a sharp tip, a rectangular rod or triangular rod. The present invention also includes embodiments where the circular conductive cylinders are replaced by rectangular conductive cylinders or cylinders with other geometrical shapes. The shapes and geometrical dimensions of the electrodes 66, 67, 68, 69 and 70 are adapted accordingly in these embodiments of the present invention.
From the geometrical dimensions of the electrodes 66 and 67 and the guard 77 in
The geometrical dimensions of the electrodes 66 and 67 and the guard 77 in
The present invention also includes embodiments with sets of multitudes of electrodes comprising circular plates and ring plates with increasing inner diameters which are larger than the outer diameter of the circular conductive plates being embedded at least partly inside. The present invention also includes embodiments, where the first electrodes as circular conductive discs 66 and 68 are replaced by circular conductive rings or other configurations like but not limited to a needle with a sharp tip, a rectangular plate or triangular plate. The present invention also includes embodiments where the circular conductive ring plates 67, 68 and 69 are replaced by rectangular conductive ring plates or plates with other geometrical shapes.
From the geometrical dimensions of the electrodes 110 and 114 and the guard 120 in
The present invention also includes embodiments with sets of multitudes of electrodes disposed on one or more foils on top of adhesive elements, the electrodes comprising circular plates and ring plates with increasing inner diameters which are larger than the outer diameter of the circular conductive plates being embedded at least partly inside. The present invention also includes embodiments, where the circular conductive disc 110 is replaced by a circular conductive ring or other configurations like but not limited to a needle with a sharp tip, a rectangular plate or triangular plate. The present invention also includes embodiments where the circular conductive ring plates 114 are replaced by rectangular conductive ring plates or plates with other geometrical shapes.
The present invention also includes embodiments where connections are made through printed circuit boards. These boards may also include switching circuitry, electronic circuitry for the impedance analysis and other circuitries to facilitate the docking, adjustments of the docking, calibration, compensation, guarding, or other purposes.
The adhesive element 118 may be a pressure sensitive adhesive based on materials such as but not limited to polyacrylates and silicone rubber and may comprise removable adhesives designed to repeatedly stick and unstick, and the foil 119 may be a polymer foil made of a polymer such as but not limited to polypropylene, polyvinyl chloride and polyethylene. The adhesive may be equipped with a release liner, which is removed prior to disposing the adhesive to the bottom plate of the microwell plate. The electrodes 110 and 114 may be screen printed or tampo printed onto the foil 119, where tampo printed is also known as pad printing and tampography, and may be of a conductive film of a material such as but not limited to silver, aluminium and gold.
In an embodiment, the first electrode 133 and/or the second electrode 135 may further comprise one or more spring elements applying a mechanical force between the top surfaces of the first electrode 133 and the second electrode 135 and outer surface of the bottom wall of the flask 134.
At the position where the first electrode 131 and the second electrode 135 are detached from the outer surface of the bottom wall of the flask 134, the docking arrangement is in a first position. At the position where the first electrode 131 and the second electrode 135 are attached to preselected locations on the outer surface of the bottom wall of the flask 134, the docking arrangement is in a second position.
The embodiment of the present invention as illustrated in
The present invention also comprises embodiments where the attachment and detachment of the first electrode 133 and the second electrode 135 to the bottom outer wall of a flask 134 are established fully or partly by means of gravity. The present invention also comprises embodiments, where the inner wall of the docking station 146 is adapted to work as a spring element ensuring tight attachment of the first electrode 133 and the second electrode 135 to the bottom outer wall of the flask 134, and where the docking station 131 is further being adapted to ensure that the flask may be repositioned reproducible at a docking site 130 with a preselected location on the outer surface of the bottom wall of the flask when detaching and attaching the flask 132 from the docking station 131.
The present invention also includes embodiments where the electrodes 133 and 135 are combined with multitudes of electrodes comprising circular plates and ring plates with increasing inner diameters, which are larger than the outer diameter of the circular conductive plates being embedded at least partly inside. The present invention also includes embodiments, where the circular conductive disc 133 is replaced by a circular conductive ring or other configurations like, but not limited to a needle with a sharp tip, a rectangular plate or triangular plate. The present invention also includes embodiments where the circular conductive ring plates including 135 are replaced by rectangular conductive ring plates or plates with annular or other geometrical shapes. The present invention further includes embodiments where the spring element 139 is engaged with an additional strip shaped electrode. Laboratory flasks may have, but are not limited to have volumes in the range from 50 mL to 3 L with a wall thickness typically in the range from 1 mm to 3 mm, but not limited to this range, and be made of a glass such as but not limited to borosilicate or soda lime, or plastics such as but not limited to polypropylene, polymethylpentene, polycarbonate or polytetrafluoroetylen. There may be variations in the sizes and diameters of the flasks used with the present embodiment of the invention. The inner wall of the docking station 146 and the diameter 140 of the spring element 139 should be designed in order to accept the tolerances or the variations in the diameters of the flasks used.
From the geometrical dimensions of the electrodes 133 and 135 in
The adhesive element 144 may be a pressure sensitive adhesive based on materials such as but not limited to polyacrylates and silicone rubber and may comprise removable adhesives designed to repeatedly stick and unstick, and the foil 145 may be a polymer foil made of a polymer such as but not limited to polypropylene, polyvinyl chloride and polyethylene. The adhesive element 144 may be equipped with a release liner, which is removed prior to disposing the adhesive below the docking station 131.
In some embodiments of the present invention, the first rail 155 and the second rail 156 in combination with the constraining element 148 ensure sufficient force on the first side of a microwell plate 158 and the second side of a microwell plate 159 to reproducibly hold the microwell plate positioned with a sufficient pressure against each set of first electrode 161 and second electrode 166. The constraining element 148 may act as a spring and is preferably at least partly made from an elastic material such as a polymer or a metal such as stainless steel.
At each container 150, by means of a first movable pin body 161 and a second movable pin body 162, a set of electrical connectors 163 and 164 physically separated from each other and shielded by a guard 165 are brought in electrical connection via a physically separated set of electrodes 161 and 166 to the insulated wall 151 of the container 150. Each set of one or more electrical connectors 163, 164 used for the microwell plate comprise a suitable termination such as, but not limited to a BNC, SMA or SMB connector.
Each set of one or more electrical connectors may be electrically connected or connectable through a first set of cables 168 to an electrical switch 169, and the switch is further electrically connected or connectable to an analyser 171 with one or more sets of channels 172 through a second set of cables 170; the electrical switch 169 being adapted to direct electrical signals from a guard 165 and from the one or more sets of electrodes 166 and 167 both having contact interfaces with the outer surfaces of insulated walls 151 of each container 150 to a set of channels 172 of the analyser 171, which derives impedance data from the microorganisms in each container and displays and/or stores the data as real time detection of growth of microorganisms in a memory of a computer or the like 173.
The analyser may be an electrochemical analyser such as an instrument making measurements of impedance parameters, like impedance magnitude and phase as a function of frequency, or components values, such as but not limited to inductance (L), capacitance (C) and resistance (R), as a function of frequency. The analyser may be, but is not limited to a commercial impedance analyser, LCR meter, vector impedance meter, LCZ meter, or a network analyser with suitable software that allows derivation of impedance features, or an instrument made from generators, digitizers and appropriate signal conditioning devices and software to calculate impedance properties, or a customized instrument made from suitable components and with software to calculate impedance properties. The connector topology of the analyser includes, but is not limited to common 4-terminal, 2-terminal or 1-terminal configurations. The instrument may apply a single tone signal, a swept signal, or a multitude of signals. The instrument may also be an instrument which operates in the time domain by applying a signal including but not limited to pulses or pulse trains, ramps, or composite signals, and which derives impedance properties through data processing. The analyser may be separate from the docking system; it may be built into the docking system, or parts of the analyser may be built into the docking system.
One embodiment of the present invention with a docking system comprising a docking station and multiple sets of electrodes adapted to analyse a sample with growth of microorganisms in multiple containers may comprise one analyser and a switching network. Another embodiment of the present invention of a docking system with a docking system comprising a docking station and multiple sets of electrodes may comprise one analyser adapted for analysis of each container. A further embodiment of the present invention may comprise multiple analysers, but fewer than the number of containers, and a switching network. The docking system, the containers and the switching network may include shielding or guarding, active or passive. The docking system may include components to calibrate the instrument and components for compensation of parasitic capacitance, like stray capacitance, unwanted inductive coupling, series inductance, series resistance or parallel conductance. The switching network may comprise an array of mechanical relays, semiconductor relays or switches, or a combination of these.
The analyser may also be a 2-terminal network analyser, which measures the electrical complex transfer function of the sample located in a container in shape of a bottle or other labware and electrically connected or connectable through the docking system to the 2 terminals of the network analyser. The electrical complex transfer function includes, but is not limited to measures of transfer magnitude and phase, or transfer scattering parameters, known as S-parameters, S12 or S21. The impedance properties of the sample in the container may be derived from the electrical complex transfer function by use of software.
The analyser may also be a 1-terminal network analyser or impedance meter, which measures the electrical complex reflection function or the impedance of the sample located in a container in shape of a bottle or other types of labware and electrically connected or connectable through the docking system to the terminal of the analyser. The electrical complex reflection function includes, but is not limited to measurements of reflection scattering parameters, known as the S-parameters, S11 or S22. The properties of the sample in the container may be derived from the electrical complex reflection function or impedance by use of software.
In addition to common scattering parameters (S-parameters) the analyser may also exploit X-parameters, which are a generalization of the linear S-parameters to characterize non-linear components and systems, and any other parameters that may be applied to characterize any non-linear properties of the sample in the container.
In one embodiment of the present invention, impedance matching is included in order to obtain larger or smaller voltages or currents of excitation or detection, or to improve the impedance matching, or for other purposes. An impedance matching may be integrated into the docking system, or may be disposed outside the docking system.
The electrical switch may comprise an array of mechanical relays with low contact resistance and low capacitance such as but not limited to the Pickering Series 103, Low Capacitance SIL/SIP Reed Relays. Alternatively, the electrical switch may comprise an array of solid-state relays or other types of relays or switches. The analyser may be an instrument making measurements in the frequency domain such as an LCR meter measuring inductance (L), capacitance (C) and resistance (R), including but not limited to a Keysight Precision LCR meter (20 Hz-2 MHz) E4980A, a Keysight E4990A impedance analyser with a frequency range from 20 Hz to 120 MHz or a Wayne Kerr 65120B Precision Impedance Analysers operating at frequencies from 20 Hz to 120 MHz; or it may be a vector network analyser such as, but not limited to a Rohde & Schwarz ZNB20 operating at frequencies from 100 kHz to 20 GHz, or other types of instruments operating in the frequency domain. When making analysis above several MHz and in particular in the GHz regime, it becomes increasingly important to match the impedance between the electrical connectors 163 and 163 with the characteristic impedance of the analyser, typically 50Ω. A person skilled in the art knows how to optimize the impedance matching.
The method of the present invention also includes steps of making open-circuit measurements, short-circuit measurements, load measurements and when required at low conductivities, low-loss capacitor measurements prior to making analysis of growth of microorganisms. Open-circuit measurements are made with an open-circuit-component docked into the docking station 153 and making impedance measurements at each preselected frequency or over the frequency range used by the analyser 171 for each set of one or more electrical connectors 163, 164 used for the microwell plate, and with the corresponding positions of the relays in the electrical switch 169. The open-circuit-component may be a microwell plate identical to or of the same type as the one used in analysis of a sample with growth of microorganisms, where each well or container is empty or filled with a gas such as air, or where each well or container is filled with a calibration fluid such as DI water, a solution of a salt such as KCl or a fluid such as a growth medium including, but not limited to BHI media. Short-circuit measurements are made with a short-circuit-component, by short circuiting each set of one or more electrical connectors 163, 164 used for the microwell plate and the guard, and making impedance measurements at each predefined frequency or over the frequency range used by the analyser. The short-circuit-component exhibits a high electric conductivity and may comprise conductive plates or wires of metals such as, but not limited to copper, gold, silver, conductive polymer films, and carbon-based films. The open-circuit measurements should preferably be made with each set of first movable pin body 161 and second movable pin body 162 essentially be at the same preselected position as when the analyses are made on a microwell plate 152 docked into the docking station 153 and with the open-circuit-component be positioned at the predefined position. The short-circuit measurements should preferably be made with each set of a first movable pin body 161 and a second movable pin body 162 essentially be at the same predefined position as when analyses are made on a microwell plate 152 docked in the docking station 153 and with the short-circuit-component be positioned at that preselected position.
Load measurements are made with a load-component having a known impedance, by connecting the load-component to each set of one or more electrical connectors 163, 164 used for the microwell plate and the guard, and making impedance measurements at each predefined frequency or over the frequency range used by the analyser. The load-component exhibits a well-known impedance or electric conductivity and may comprise passive and/or active components. Preferably, the value of the load should be within the measurement range of analyser. In case the analyser has multiple measurement ranges, different values of load can be used, and measurements are carried out for each range to establish traceability. A person skilled in the art knows how to select the load impedance values and to carry out load measurements. The load measurements should preferably be made with each set of first movable pin body 161 and second movable pin body 162 essentially be at the same preselected position as when the analyses are made on a microwell plate 152 docked into the docking station 153 and with the load-component be positioned at the predefined position.
The method of the present invention also includes steps of modelling the impedance data by equivalent electrical circuits. A number of models have been reported in the scientific literature, including [C. E. Turick et al. Appl. Microbiol. Biotechnol. 103, 8327-8338 (2019)], [R. Gnaim et al. BioTechniques 69, 27-36 (2020)], [A. L. A. de Araujo et al. Biosensors 9, 108, 1-4 (2019)] and [M. Simić et al. IEEE Sensors J. 20, 12791-12797 (2020)]. The equivalent electrical circuits modelling the embodiments of the present invention include a capacitor related to the electrically insulated walls of containers and should be included in the equivalent electrical circuit as a parallel capacitor. The modelling may determine the species of the microorganisms and discriminate dead microorganisms from live microorganisms.
Simple circuit models may be expressed in terms of parallel admittance (Yp), where a conductance (Gp or the reciprocal resistance, Rp) and a susceptance (Bp or the reciprocal reactance, Xp) are connected in parallel. Alternatively, simple circuit models may be or expressed in terms of series impedance (Zs), where a resistor (Rs) and a reactance (Xs) are connected in series. There are several other combinations of making circuit models, and it is simple to transform one circuit model into another type of circuit model. The relationship between (Rp, Bp) and (Rs, Xs) is;
The method of the present invention also includes steps relating the impedance data as detected by the analyser 171 to a number of microorganisms, which includes taking subsamples from the sample of microorganisms 154 and count the number of microorganisms using standard methods such as colony-forming-units (CFU), optical density measurements at 600 nm (OD600) using calibrated instruments, or using similar methods. A person skilled in the art knows how to make such calibration measurements. Making open-circuit measurements and short-circuit measurements subtracts the effect of the electrically insulated walls of a container, which can be considered to be a parallel capacitor. In combination with reproducible positioning of each container used of a microwell plate at essentially the same preselected location when attaching and detaching the microwell plate ensures reproducible measurements and enable absolute detection of the number of microorganisms in real time.
In an embodiment, the first electrode 186 and/or the second electrode 187 may further comprise one or more spring elements applying a mechanical force between the top surfaces of the first electrode 186 and the second electrode 187 and the outer surface of the bottom wall of the flask 185.
The first, the second and the third electrical connectors 188, 189 and 191 are electrically connected or connectable through a set of cables 192 to an analyser 193 by directing electrical signals from the guard 190, each set of first electrode 186 and second electrode 187 having contact interfaces with the insulated outer surface of the bottom wall of the flask 185 to the analyser 193, which derives impedance data from the microorganisms in the flask and displays and/or stores the data of real time detection of growth of microorganisms in a memory of a computer or the like 194.
The docking system may further comprise an adhesive element 195 and a foil 196, where the first electrode 186, the second electrode 187 and the guard 190 are disposed on top of the foil 196. The electrodes 186 and 187 may be screen printed or tampo printed onto the foil 196 and may be of a conductive film of a material such as, but not limited to silver, aluminium, copper of gold. The present method of the invention may also include attaching the adhesive element 195, the other parts of the docking system and the flask 181 in an orbital shaker in order to ensure effective stirring of the fluid comprising the microorganisms.
The method of the present invention also includes steps of making open-circuit measurements and short-circuits measurements prior to making analysis of growth of microorganisms. Open-circuit measurements are made with an open-circuit-component docked into the docking station 183 and making impedance measurements at each preselected frequency or over the frequency range used by the analyser between the electrical connectors 188 and 189. The open-circuit-component may be a flask identical to or of the same type as the one used in analyses of growth of microorganisms, where the flask is empty or filled with a gas such as air, or where the flask is filled with a calibration fluid such as water, a solution of a salt such as KCl, or a fluid such as a growth medium including but not limited to BHI media.
In combination with reproducibly docking the container into a docking station with attachment and detachment of the containers at preselected locations of electrodes on the outer surface of one or more containers and with reproducible tightening pressure at the contact interfaces between the electrodes and the outer walls of the containers, the open-circuit-measurement enables elimination of individual variations in container wall thickness, variation in container wall material composition, wall roughness and other materials and structural parameters.
Short-circuit measurements are made with a short-circuit-component by short circuiting the electrical connectors 188 and 189 and the electrical connector for the guard 191 and making impedance measurements at each preselected frequency or over the frequency range used by the analyser. The short-circuit-component exhibits high electric conductivity and comprises one or more conductive plates or wires of conductive materials such as, but not limited to copper, gold, silver, conductive polymer films, carbon-based films or the like. The open-circuit measurements should preferably be made with the first electrode 186 and the second electrode 187 essentially at the same preselected location as when the analyses are made on a flask 181 docked into the docking station 183 at the docking site 180 and with the open-circuit-component be positioned at that preselected location. The short-circuit measurements should preferably be made with the first electrode 186 and the second electrode 187 essentially be at the same predefined positions as when the analyses are made on a flask 181 docked into the docking station 183 at the docking site 180 and with the short-circuit-component be positioned at the preselected location.
Open-circuit measurements and short-circuit measurements are procedures, which are normally integrated in analysers such as LCR-meters and impedance analysers. For an analyser with no integration of such procedures, a person skilled in the art knows how to correct the impedance data recorded with the analyser with respect to the open-circuit measurements, the short-circuit measurements, and the load measurements.
The method of the present invention also includes analysers based on measurements in the time domain short as methods based on applying current pulses and measuring the voltage response, or applying voltage pulses and measuring the current response. The width of the pulses, repetition rates and duty cycles used for the analysers making measurements in the time domain can be optimized according to knowledge of suitable frequencies used with analysers making measurements in the frequency domain and using mathematical tools such as Laplace transforms or inverse Laplace transforms, which relate the frequency domain and the time domain. This is described in the literature, e.g. in “C. Thirstrup and L. Deleebeeck, IEEE Trans Instrum Meas 70, 1008222 (2021)”.
A docking system with non-contact electrodes adapted for real time detection of growth of microorganisms through insulated walls of a container as illustrated schematically in
The low current and low potential output from the LCR meter were connected to the first electrical connector 34, and the high current and high potential output from the LCR meter were connected to the third electrical connector 36 via BNC cables through an electrical switch based on the Pickering Series 103, Low Capacitance SIL/SIP Reed Relays. The guard of the LCR meter was connected to the fourth electrical connector 37 and to the second electrical connector 35 via BNC cables through the electrical switch. Prior to the measurements on microorganisms, open-circuit measurements for the LCR meter were made with a 500 mL glass bottle docked into the docking station 21 and the docking screws 31 and 32 were tightened using a torque wrench with specified torque of 20 Nm; short-circuit measurements were made connecting the first electrical connector 34, the second electrical connector 35 and the third electrical connector 36 to the fourth electrical connector 37 connected to the guard of the LCR meter.
The bacteria S. epidermidis were grown in BHI media in the 500 mL glass bottle at 37° C. in a temperature stabilized air bath. A Teflon coated magnet (ø8.0 mm×30.0 mm) was immersed at the bottom of the bottle; the bottle was docked into the docking station 21 and a magnetic stirrer (IKA model RCT classic) was mounted below the docking station 21 with a stirring speed set to 200 rpm. The docking screws 31 and 32 were tightened using a torque wrench with specified torque of 20 Nm. Data of parallel impedance (change in real part of the parallel impedance, resistance (ΔRp) and change in the imaginary part of the parallel admittance, susceptance (Bp)) from the LCR meter were made in the frequency range from 100 Hz to 2 MHz with a sweep of eighty selected frequencies and at a voltage of 1 V.
The data of non-contact electrochemical impedance (Rp, Bp) of bacterial growth recorded as function of time were fitted by sigmoid functions (“s”-curves) according to;
where U=ΔRp for data of parallel resistance in units of Ω, and U=ΔBp for data of parallel susceptance in units of S; ΔUs is the data for t=+∞; τ is an exponential time constant; and t0 is the time at the onset of the exponential bacterial growth. The model in Eq. (2) includes a lag phase, an exponential growth phase, and a stationary phase (M. Peleg and M. G. Corradini, Critical Rev. Food Sci. Nutr., vol. 51, pp. 917-945, 2011).
For S. epidermidis at 37° C., data of changes in the real part of the impedance (ΔRp) recorded between electrode 22 and electrode 27 at 10 kHz are plotted as solid curve as function of time (t) in
As observed in
A docking system 41 being adapted to dock with non-contact electrodes adapted for real time detection of growth of microorganisms through insulated walls of a container as illustrated schematically in
The electrodes were screen printed with conductive silver ink on top of a thin foil with an adhesive on the distal surface of the foil. The first electrode was a 4 mm disc surrounded by a second electrode comprising a ring configuration with an inner diameter of 8 mm and an outer diameter of 14 mm.
S. epidermidis bacteria were grown in BHI media in a microwell at 37° C. in a temperature stabilized air bath. Data of series impedance (change in real part of the series impedance, resistance (ΔRs) and change in the imaginary part of the series reactance (−ΔXs, see Eq. (1)) from the impedance analyser were made in the frequency range from 1 MHz to 1 GHz with a sweep of eighty selected frequencies and at a voltage of 1 V.
For S. epidermidis bacteria grown in BHI media at 37° C., data of changes in the real part of the impedance (ΔRs) recorded between electrode 110 and electrode 114 at 10 MHz are plotted as solid curve as function of time (t) in
The dot-dashed curve in
In
| Number | Date | Country | Kind |
|---|---|---|---|
| 2022 00590 | Jun 2022 | DK | national |
