Patent Description:
Living cells typically consume oxygen (O2) from their surrounding environment and release metabolic byproducts, such as carbon dioxide (CO2), lactate, and various other metabolic byproducts. Flux analyzers allow one to measure the oxygen consumption rate (OCR), extracellular acidification rate (ECAR), CO2 production rate (CPR), and/or other biological flux parameters. Such measurements can provide valuable information regarding the metabolic processes carried out by these living cells.

One known method of measuring the OCR and ECAR of living cells is by measuring flux of O2 and H+ generated by these living cells in a wellplate using a set of fluorescent sensors. An example of an analyzer using fluorescent sensors to detect flux of O2 and H+ is the Seahorse flux analyzer, which is generally described in <CIT> and <CIT>. The fluorescent sensors measure the intensity of fluorescent signals over time. For a sample containing living cells, those signals over time will be proportional to the rate of production or consumption of O2 molecules and H+ ions consumed or produced by the metabolism of the cells. This data is used to calculate the flux of O2 molecules and H+ ions consumed and produced by the living cells.

Problems connected with flux analyzers and flux measurements include a lack of standardization and unavailability of precise calibration references. Because flux is measured over time rather than at steady state conditions, the rate of chemical diffusion or permeation into the sensor and surrounding medium can vary between analytes creating a complex and dynamic measurement environment. Currently, to address these problems, flux measurement systems typically apply complicated algorithms to compensate for the various rate constants associated with each analyte. These rate constants are empirically derived based on biological references. Final verification of system performance is commonly done using biological standards that contain a known content of cells of a certain type; for example, a well characterized, immortalized, rodent, skeletal muscle fibroblast cell line (C2C12) is frequently used as a standard. These cell lines are maintained in culture in a level <NUM> biolab prior to use. In preparation for the assay the cells are trypsinized, counted and seeded into a well plate. The seeding quantity is controlled so that <NUM> hrs post seeding the cell population has grown to a measureable confluencey and is ready for use in the instrument. Because the cells continue to grow/expand after seeding the timing of the assay is critical to attain reproducible results. In addition, cell lines are inherently variable and subject to variability caused by passage, genotype, culture conditions. Because of these variables the typical reproducibility between wells, between assays often exceeds <NUM>% cv. The variability of biological standards diminishes their capability of being suitable standards to use for verifying the performance of the flux measurement system.

Other methods for generating flux within a wellplate include the use of enzymes/catalysts such as glucose oxidase (GOx), sodium sulphate, or redox reactions. These methods have been previously used but are also subject to variability and complexity. For example; a typical enzymatic reaction is dependent on two or more compounds reacting to generate the desired signal. The reactions of the two compounds is concentration dependent, thus flux is variable (nonlinear) as the catalyst is consumed as part of the reaction. Additionally, these methods are difficult to tune in terms of a rate constants and become complex when more than one flux is desired. For such a method to replicate biological activity, reaction components would need to be adhered to the bottom of the plate, be linearly catabolized, at a controllable rate. These needs make this is a difficult and complex assay. <CIT> discloses a dialysis apparatus including a multiwell plate), a separation membrane and a template having holes which mate with the wells. The membrane is sealed to one end of the wells by positioning the template about the outer peripheral surfaces of the wells with the membrane being frictionally held therebetween. <CIT> discloses a system for measuring diffusion of a compound across a membrane that comprises a membrane held between a first base having a plurality of outwardly extending hollow projections and a second base fastened to the first base and having a plurality of recessed tapered openings therein adapted to engage the plurality of hollow projections. <CIT> relates to methods for producing island-shaped three-dimensional cell aggregates that enable optical image analyses with increased analytical robustness, amenable to high through-put analyses for drug screening, toxicological assays, drug screening, and/or high content analyses of the milieu of other cytological events. <CIT> discloses an apparatus for analysis of cells disposed in media in multiple wells of a multi-well plate, the comprises: a plurality of barriers for insertion into respective wells of the multi-well plate, each barrier comprising a barrier surface that creates, when inserted into the respective well, a sample chamber having a reduced volume of medium less than <NUM>% of the original volume of medium in the wells, and disposed on barrier surfaces, fluorescent sensors for analyzing a constituent of the medium disposed about the cells in the respective sample chamber. <CIT> relates to measurements of the acidification rate and oxygen consumption rate of an extracellular medium surrounding cells and discloses an apparatus for measuring extracellular acidification rate or CO <NUM> evolution of a cell culture disposed in a medium, the apparatus comprising a compartment defining an interior surface and a volume for holding cells disposed within a cell medium. <CIT> discloses a multiple well tissue culture plate having a plurality of opened bottomed wells with a semi-permeable membrane across the bottoms of a plurality of such wells. The multiple well tissue culture plate is placed in contact with the basal medium contained within the basal medium reservoir to allow for exchange of nutrients and waste products between the basal medium and the culture medium across the semi-permeable membrane.

In summary, there is a need for better standards such as devices that do not rely on cells to be used to calibrate the performance of a flux analyzer.

Any "embodiment" or "example" which is disclosed in the description but not covered by the claims should be considered as presented for illustrative purpose only. The term "flux" as used herein means change of a parameter as a function of time. The change may be expected due to consumption or reaction of a reactant represented by the parameter.

Referring now to <FIG> and <FIG>, the flux block of the present disclosure is generally indicated by numeral <NUM>. The flux block <NUM> includes: (<NUM>) a first frame <NUM>; (<NUM>) a second frame <NUM>; (<NUM>) and a selectively permeable membrane <NUM>. In this embodiment, the first frame <NUM> of the flux block <NUM> and the second frame <NUM> of the flux block <NUM> are connected or integrally formed. For example, the flux block <NUM> can be formed from two or more separately formed sections (such as halves) that are suitably connected. The frames can have physical mating surfaces that permanently or removably interlock, such as where the edges snap together. Alternatively, the frames can be connected to each other by an adhesive or by soldering together.

More specifically, the first frame <NUM> includes a plurality of walls, including: (<NUM>) a first wall <NUM>; (<NUM>) a second wall <NUM>; (<NUM>) a third wall <NUM>; and (<NUM>) a fourth wall (not shown in cross-sectional view). The walls of the first frame <NUM> include inner and outer surfaces. In certain embodiments, the first frame <NUM> defines a multi-well microplate or well plate. The plurality of walls of the first frame <NUM> partially encloses a plurality of wells <NUM>. In certain embodiments, the plurality of wells <NUM> of the first frame <NUM> is disposed in four different columns and six different rows. Thus, the first frame <NUM> in this embodiment comprises twenty-four wells. The plurality of wells <NUM> can be conical or partially conical.

In certain embodiments, the plurality of wells <NUM> of the first frame <NUM> can be disposed in a different number of columns, a different number of rows, or a combination thereof. For example, another embodiment of the flux block <NUM> of the present disclosure can include a well plate including a plurality of wells <NUM> that are disposed in eight different columns and twelve different rows. Thus, this alternative embodiment of the well plate can include ninety-six wells. Yet another embodiment of the flux block comprises a single row of wells, for example, a single row of eight, <NUM>, <NUM> or <NUM> wells. Yet another embodiment of the flux block comprises <NUM> different columns and <NUM> different rows. Thus, this alternative embodiment of the well plate can include <NUM> wells.

<FIG> illustrates a closer view of a single well <NUM> of the first frame <NUM> of the flux block <NUM>, as well as its relation to a membrane <NUM> and a second frame <NUM>. The walls <NUM> of this well <NUM> define a well void <NUM> configured to hold a volume of a suitable liquid sample(s). The walls <NUM> of the well <NUM> include inner and outer surfaces. The well <NUM> is configured to receive a plunger or other sensor, as further described below.

The flux block <NUM> of the present disclosure can include a filler <NUM>. It should be appreciated that the filler <NUM> can be optional. The filler <NUM> can be made of the same material as the first frame <NUM> and/or the second frame <NUM>. The filler <NUM> can also be made of other suitable material. The filler <NUM> should not interfere with the operation of the flux block <NUM>.

The well <NUM> includes a well base <NUM>. The well base <NUM> of the well <NUM> includes a first surface <NUM> and a second surface <NUM>. The second surface <NUM> engages a first separator <NUM>. The well base <NUM> and the first separator <NUM> each has an opening, and together they define a well opening <NUM>. The well opening <NUM> allows CO2, O2, and/or other gas or liquid molecules to move in and/or out of the well void <NUM> of the well <NUM>. In certain embodiments, liquid and or solid molecules can move through the well opening <NUM> to further move in and/or out of the well void <NUM> of the well <NUM>.

In certain embodiments, the first surface <NUM> of the well base <NUM> has an uneven surface, and might be slightly raised in its outer perimeter than its inner perimeter. In certain embodiments, a sensor stop <NUM> extends from the first surface <NUM> of the well base <NUM> of the well <NUM>. The sensor stop <NUM> is configured to engage a plunger when the plunger is seconded into the well void <NUM> of the well <NUM>. Altematively or additionally, a raised portion of first surface <NUM> may engage a sensor or plunger so as to prevent it from contacting a lower portion. More details regarding the use of the sensor/plunger are discussed below. In certain embodiments, the flux block <NUM> of the present disclosure can include one or more sensor stops <NUM> connected to and extending from the first surface <NUM> of the well base <NUM> of the well <NUM>. In certain embodiments, the one or more sensor stop <NUM> can be defined by the inner surfaces of the well walls <NUM> of the wells <NUM>. The one or more sensor stop <NUM> can be angled or sloped. Certain embodiments of the flux block <NUM> of the present disclosure can include one or more stop sensor <NUM> that lie in a step-like manner rather than being sloped or angled. Thus, the top surfaces of the one or more sensor stop <NUM> can be parallel to the top surface <NUM> of the well base <NUM> of the well <NUM>.

Each of the wells may have a top portion with an opening having an area A1 as well as a bottom portion that may be cylindrical or square having an area A2, and the well may be defined by a tapered sidewall. A2 can be significant smaller than A1. A seating surface may be provided to act as a positive stop for sensors disposed on barriers. This seating surface enables the creation of a localized reduced volume of medium, as discussed in <CIT>. In an embodiment, the seating surface may be defined by a plurality of raised dots, e.g., three dots, on a bottom surface of a well. The well areas and depths can be any dimension and may be preferably selected from an area A1 of <NUM> to <NUM><NUM>, alternatively <NUM><NUM>, an area A2 of <NUM> to <NUM><NUM>, alternatively <NUM><NUM> , and a depth of the wells may range from <NUM> to <NUM> or more, preferably about <NUM>; embodiments having those dimensions include plates comprise a single column or row of wells, and plates comprising <NUM> or <NUM> wells. In another embodiment, where the plate comprises <NUM> wells, and the well areas A1 and A2 and depths are the same as above, or proportional to the above numbers. Preferably, the wells are spaced equally from each other. Each of the wells in the microwell plate can have substantially the same dimensions or can have varying dimensions.

The second frame <NUM> includes a plurality of walls, including: (<NUM>) a first wall <NUM>; (<NUM>) a second wall (not visible); (<NUM>) a third wall <NUM>; (<NUM>) a fourth wall (not shown); (<NUM>) a fifth wall <NUM>; and (<NUM>) a sixth wall <NUM>. In this embodiment, the first wall <NUM> defines an inlet/outlet port <NUM> configured to receive a tube (not shown). It should be appreciated that certain embodiments of the flux block <NUM> of the present disclosure can include other suitable walls of the second frame <NUM> that define the inlet/outlet port <NUM>. The walls of the second frame <NUM> include inner and outer surfaces. The sixth wall <NUM> can be removed to clean the flux block <NUM>. The sixth wall <NUM> is removable from the second frame for suitable purposes such as clearing or repair.

In certain embodiments, the walls of the second frame <NUM> define a chamber <NUM> configured to hold a volume of suitable substance, such as CO2, O2, or a combination thereof. It should be appreciated that the chamber <NUM> can hold a volume of other substances, and the chamber can be adapted for holding such substances, such as by having a coating on its surfaces. It should further be appreciated that certain embodiments of the flux block <NUM> of the present disclosure can include walls of the second frame <NUM> that define a chamber <NUM> configured to hold a volume of suitable liquid sample(s), other suitable gaseous sample(s), or a combination thereof. The chamber can be divided into a plurality of subchambers and the frame can have different inlet-outlet parts for different subchambers.

The first surface <NUM> of the top wall <NUM> engages the second separator <NUM>. The top wall <NUM> and the second separator <NUM>, if present, define a chamber opening <NUM> that is aligned with the well opening <NUM>. The chamber opening <NUM> and the well opening <NUM> are sufficiently aligned or concentric to allow CO2, O2, and/or other molecules to move in and/or out of the chamber <NUM> of the second frame <NUM> and into the well void <NUM>.

The selectively permeable membrane <NUM> is configured to allow one or more substances to permeate across the permeable membrane <NUM>, where those substances constitute or cause a flux which is measured by a flux analyzer. For example, the membrane may permit CO2, O2 or both to pass from one side of the membrane to the other. The selectively permeable membrane also acts as a barrier to one or more of water, other solvents, and other molecules. Generally, the permeable membrane prevents bulk movement of liquid in the wells into the chamber. As illustrated in <FIG>, the selectively permeable membrane <NUM> is held between the first separator <NUM> and the second separator <NUM>. The first separator <NUM> and second separator <NUM> are configured such that the permeable membrane <NUM> does not generally move when positioned between the second surface <NUM> of the well base <NUM> of the well <NUM> and the first surface <NUM> of the fifth wall <NUM> of the second frame <NUM>. The permeable membrane <NUM> acts as a boundary between the well voids <NUM> of the wells <NUM> and the chamber <NUM> of the second frame <NUM>.

In certain embodiments of the flux block <NUM>, the first separator <NUM> and the second separator <NUM> are configured such that when positioned between the first frame <NUM> and the second frame <NUM>, the permeable membrane <NUM> is equidistant from the first surface <NUM> of the top wall <NUM> of the second frame <NUM> and the second surface <NUM> of the well base <NUM> of the well <NUM>. In certain embodiments of the flux block <NUM> of the present disclosure, the first separator <NUM> and the second separator <NUM> can be configured such that when positioned between the first frame <NUM> and the second frame <NUM>, the permeable membrane <NUM> is positioned other suitable distances from the first surface <NUM> of the top wall <NUM> of the second frame <NUM> to the second surface <NUM> of the well base <NUM> of the well <NUM>. The separators and frames can have alignment indicia to ensure proper alignment. Furthermore, the permeable membrane <NUM> is a boundary between the well voids <NUM> of the wells <NUM> and the chamber <NUM> of the second frame <NUM>.

In certain embodiments, the flux block <NUM> of the present disclosure does not include the first separator <NUM> and the second separator <NUM>. In such embodiments, the selectively permeable membrane <NUM> engages the first surface <NUM> of the top wall <NUM> of the second frame <NUM> and the second surface <NUM> of the well base <NUM> of the well <NUM>. In certain embodiments, the membrane extends across the face of the second frame <NUM> closest to the first frame <NUM>. In such embodiments, the membrane <NUM> is a boundary between the well voids <NUM> of the wells <NUM> and the chamber <NUM> of the second frame <NUM>. It should further be appreciated that the membrane <NUM> can be integral with either or both of the first frame <NUM> and the second frame <NUM>. The membrane can be a single sheet that extends over substantially the whole surface of the first frame and/or second frame, or it can be in discrete patches that cover the well openings.

The flux block <NUM> of the present disclosure can be formed from a molded plastic, such as polystyrene, polypropylene, polycarbonate, or other suitable material. The bottoms of the wells may be opaque or reflective so that a sensor in the well collects light emitted or reflected by the contents in the well, or the well bottoms may be transparent to allow light to pass through those bottoms. In certain embodiments, the sides of the wells are opaque to reduce optical cross-talk or light contamination from one well to another. In some embodiments, e.g., for use with luminescence measurements, the wells may be white or reflective.

In certain embodiments, the permeable membrane <NUM> is a polymethylpentene film, such as a TPX film. Alternatively, the permeable membrane <NUM> can be polystyrene. It should further be appreciated that certain embodiments can include a selectively permeable membrane made of other suitable materials. In some embodiments, the membrane is removable from the apparatus, so that it can be replaced, thereby rejuvenating the calibration apparatus.

The flux block <NUM> of the present disclosure is designed for use in calibrating a well-based analytical system running a calibration application, such as an optical system or a flux analyzer. In some embodiments, a method of using the flux block of the present disclosure to calibrate a flux analyzer includes: filling the chamber of the second frame with a known CO2 concentration, such as <NUM>%; filling the well voids of the wells of the first frame with a buffer solution having a known CO2 concentration, such as <NUM>%; and measuring the change in concentration of an analyte such as CO2 or O2 or hydrogen ions over a period of time. In some embodiments, the method further comprises assessing whether the flux analyzer passed or failed. In some embodiments, the method further comprises comparing the measurements to one or more criteria and determining if the measurements meet the criteria; in those embodiments, the criteria can be a single value or a range of values. In some embodiments, the method further comprises assessing applying a correction factor to the analyzer or for further calibration.

More specifically, the method can comprise connecting a tube (not shown) to the inlet/outlet port <NUM> of the first wall <NUM> of the second frame <NUM>. CO2 molecules travel through the tube and into the chamber <NUM> of the second frame <NUM>, thus purging the chamber <NUM> of the second frame <NUM> with <NUM>% CO2. In other words, the partial pressure of CO2 in the chamber <NUM> is <NUM>%. The partial pressure of O2 in the chamber <NUM> of the second frame <NUM> is <NUM>%. Upon purging the chamber <NUM> with <NUM>% CO2, the tubing apparatus is removed from the inlet/outlet port <NUM>. A seal (not shown) covers the inlet/outlet port <NUM> by engaging the outer surface of the first wall <NUM> of the second frame <NUM>. The seal allows the chamber <NUM> of the second frame <NUM> to contain the CO2 molecules so that no CO2 leaves the chamber <NUM> and that no other gaseous or liquid sample(s) enter the chamber <NUM>.

The method further includes filling the well voids <NUM> of the plurality of wells <NUM> of the first frame <NUM> with a suitable volume of a test solution, such as a buffer solution having a pH of <NUM>. The well voids <NUM> can hold a volume of other suitable liquid sample(s) and/or a volume of media about live cells. The method further includes measuring changes or flux in one or more parameters such as CO2 while allowing the buffer solution to equilibrate under ambient conditions. The ambient conditions generally include conditions of partial pressures of gases being approximately <NUM>% O2 and <NUM>% CO2. By equilibrating under ambient conditions, the buffer solution equilibrates to include a partial pressure of CO2 that is less than the <NUM>% partial pressure of CO2 in the chamber <NUM> of the second frame <NUM>. This difference in partial pressure of CO2 creates a concentration gradient of CO2 across the permeable membrane <NUM>. Thus, so long as there is a greater concentration of CO2 in the chamber <NUM> of the second frame <NUM> than there is in the well voids <NUM> of the wells <NUM> that include the buffer solution, CO2 will continue to move across the membrane. Furthermore, while the buffer solution equilibrates under ambient conditions, a concentration gradient of O2 forms. Thus, there is a greater concentration of O2 in the well voids <NUM> of the wells <NUM> that include the buffer solution than there is in the chamber <NUM> of the second frame <NUM> that includes a <NUM>% partial pressure of O2.

When the concentration gradients described above form between the chamber <NUM> of the second frame <NUM> and the well voids <NUM> of the wells <NUM> of the first frame <NUM>, O2 molecules and CO2 molecules move down their concentration gradients. In other words, the molecules of each move from an area of higher concentration to a second concentration. In such case, the CO2 moves from the chamber <NUM> of the second frame <NUM> to the well voids <NUM> of the wells <NUM>. More specifically, CO2 molecules move through the chamber opening <NUM>. The CO2 molecules further permeate across the permeable membrane <NUM>. The CO2 molecules further travel through the well opening <NUM> of the well <NUM>, where the CO2 molecules further solubilize in the buffer solution in the well voids <NUM> of the wells <NUM> of the first frame <NUM>. The O2 molecules move from the well voids <NUM> of the wells <NUM> to the chamber <NUM> of the second frame <NUM>. More specifically, O2 molecules move through the well opening <NUM> of the well voids <NUM> of the wells <NUM>. The O2 molecules further permeate across the permeable membrane <NUM>. The O2 molecules further travel through the chamber opening <NUM> of the chamber <NUM> of the second frame <NUM> and fill the chamber <NUM>, thus increasing the O2 partial pressure in the chamber <NUM> of the second frame <NUM>. The changes or flux of the O2 molecules and the CO2 molecules in the wells during the time period of equilibration can be calculated with a high degree of confidence, as explained in more detail below. Accordingly, the concentration of O2 molecules and CO2 molecules can be measured during equilibration, and the measured values can be compared to calculated values, and the accuracy of the analyzer can be determined. If the measured values are different from the calculated values, corrective action can be taken. The corrective action may be adjusting the analyzer, applying a correction factor to the existing measured values and/or future measured values, or cleaning or replacing sensors. In this way, the analyzer is calibrated.

As CO2 molecules solubilize in the buffer solution, the CO2 molecules undergo the following reaction:.

In other words, the CO2 molecules react with the water (H2O) molecules in the buffer solution to form hydrogen ions or H+ ions. The formation of H+ ions causes the pH of the solution to decrease.

In certain embodiments, the method of measuring the change in concentration of O2 molecules and H+ ions and further calculating the flux of each analyte includes sending a plunger <NUM> into the well voids <NUM> of the wells <NUM> of the first frame <NUM>. The plunger <NUM> comprises (e.g., contains, holds or supports) one or more sensors <NUM>. <FIG> illustrates an embodiment of this process and apparatus in one well void <NUM> of a first well <NUM> of the first frame <NUM>. In this embodiment, the permeable membrane <NUM> engages the first separator <NUM> and the second separator <NUM>.

The plunger <NUM> extends from a manual or automatic actuator or other support. A barrier <NUM> is disposed at one end of the plunger <NUM>. One or more sensors <NUM> are disposed at the distal end of the barrier <NUM> of the plunger <NUM>. An example of a sensor is a fluorescent indicator, such as an oxygen-quenched fluorophore, embedded in an oxygen permeable substance, such as silicone rubber. The fluorescent indicator provides a fluorescent signal which is dependent on the presence and/ or concentration of a constituent in the well. Other types of known sensors may be used, such as electrochemical sensors, ISFET sensors, and amperametric sensors such as the Clark electrode, for example.

In certain preferred embodiments, the one or more optical sensors include fluorescent sensors. The fluorescent sensors are configured to detect the intensity of fluorescent signals that are proportional to the rate of change in concentration of O2 molecules and H+ ions in the buffer solution over a period a time. The one or more sensors <NUM> communicate electrically via fiber optics or wires <NUM> with a system <NUM> that includes a processor running a calibration procedure. The calibration procedure is programmed to calibrate the system <NUM>, using data (such as measured flux) collected, measured, or determined by the one or more sensors <NUM> in sensing communication with the analytes in the buffer solution in the well void <NUM> of the well <NUM>.

In some embodiments, upon automated actuation, plungers <NUM> descend into the well voids <NUM> of the first frame <NUM>. A single plunger descends into a given well, and the plungers can be precisely spaced so as to align with the locations of wells in a standard well plate, such as a standard <NUM>-well plate, <NUM>-well plate, or <NUM>-well plate. The barrier <NUM> of the plunger <NUM> engages the sensor stop <NUM>. Engagement of the barrier <NUM> of the plunger <NUM> and the plunger stop <NUM> of the well <NUM> defines a micro-well <NUM> having a reduced volume of media. In this embodiment, the micro-well <NUM> holds a reduced volume of buffer solution carrying out the chemical reaction(s) described above or other reactions. In some embodiments of the flux block <NUM> of the present disclosure, the micro-well <NUM> is configured to hold a volume of less than <NUM> microliters of buffer solution. This volume may be defined by the area of the bottom of the wells (for example, <NUM><NUM>) and the height of the stop barriers, for example, <NUM>). In certain embodiments, the micro-well <NUM> can be configured to hold a volume greater than <NUM> microliters of buffer solution or other suitable liquid sample(s). The micro-well <NUM> of this volume allows the one or more sensors <NUM> to measure the change in concentration of the O2 molecules and H+ ions over a period of time as O2 molecules move from the micro-well <NUM> to the chamber <NUM> of the second frame <NUM> and CO2 molecules move from the chamber <NUM> of the second frame <NUM> to the micro-well <NUM> to subsequently undergo the chemical reaction described above. The micro-well <NUM> of this volume further enhances measuring sensitivity. It should be appreciated that the micro-well <NUM> can be defined by moving the first frame <NUM> toward a stationary plunger such that the barrier of the stationary plunger or other support or arrangement of the sensor engages the sensor stops of the well <NUM> of the first frame <NUM>. The sensor stop is optional, and any arrangement where the sensor is positioned at a desired height above the sample may be used.

Upon defining the micro-well <NUM>, the one or more optical sensors <NUM> detects changes in fluorescent signals that are proportional to the rate of change in concentration of the O2 molecules and H+ ions in the micro-well <NUM> of the well <NUM> over a period of time. The one or more sensors <NUM> send data representing the change in these concentrations to the analyzer system <NUM> via the wires or fiber optics <NUM>. The processor running the calibration application receives this data and measures the flux of each analyte. The processor further calibrates the optical analyzer <NUM> using: (<NUM>) the measured flux rate of each analyte; and (<NUM>) a correction factor programmed into the calibration procedure. Further detail on the use of a plunger is provided in <CIT>.

In certain embodiments, the diameter of the wells <NUM>, and particularly the diameter D2 (as shown in <FIG>) at the bottom of the wells <NUM>, of the flux block <NUM> of the present disclosure is slightly smaller than the diameter D1 (as shown in <FIG>) of the barrier <NUM> of the plunger <NUM> such that the barrier <NUM> defines a boundary between the volume of the micro-well <NUM> and the remaining volume of the well void <NUM> of the well <NUM> when the barrier <NUM> engages the stop barrier <NUM> of the well <NUM>.

In certain embodiments, the flux block <NUM> of the present disclosure can be configured to maintain a desired flux rate (Q) over a period of time of each analyte as each analyte moves across the permeable membrane. More specifically, the variables in the following equation can be adjusted such that a desired flux rate of each analyte is maintained: (<NUM>) a thickness of the permeable membrane (t); (<NUM>) a surface area of the permeable membrane (A); (<NUM>) a differential concentration of gas and/or liquid samples across the permeable membrane (dP1); (<NUM>) a gas pressure of the gas sample in the chamber of the second frame (dP2); and (<NUM>) a permeation coefficient of the permeable membrane (K). These variables can be adjusted and used in the following equation to maintain a desired flux rate of each analyte that moves across the permeable membrane.

In certain embodiments of the flux block <NUM> and calibration method of the present disclosure, the desired flux rate for O2 molecules is approximately <NUM>-<NUM> picomoles/min. Membrane parameters, such as the thickness and the material from which the membrane is made, may be selected so that the membrane will have a desired flux rate under its contemplated conditions of use (which include the pressure of test fluid (dP1) and partial pressure of the substance such as CO2 (dP2) which will pass through the membrane at the desired flux rate). dP1 is in units of psi and dP2 is a percentage. For a membrane of polymethylpentene material, contemplated for use in a flux block where the test fluid is <NUM>% CO2 gas, the thickness will generally range from <NUM> to <NUM> and the area will generally range from <NUM><NUM> to <NUM><NUM>. For a membrane of polystyrene under the same conditions, the thickness will generally range from <NUM> to <NUM> and the area will generally range from <NUM><NUM> to <NUM><NUM>. The area of the well openings may be selected so as to determine the area of the membrane.

Certain embodiments of the flux block <NUM> of the present disclosure can be configured to allow the delivery of carbon sources, such as glucose, amino acids, fats, and active agents into the chamber <NUM> of the second frame <NUM>, the well void <NUM> of the well <NUM>, the sub-well <NUM> of the well <NUM>, or a combination thereof. It should further be appreciated that certain embodiments can be configured and/or used to control environmental conditions of a bioreactor, such as the partial pressure of O2, the pH, etc. of liquid and/or gaseous samples.

Certain embodiments of the flux block <NUM> of the present disclosure can be configured to measure the flux rate of other suitable analytes, such as a dissolved gas (e.g., O2 , CO2 , NH3), an ion (e.g., H+, Na+, K+, Ca++), a protein (e.g., cytokines, insulin, chemokines, hormones, antibodies), a substrate (e.g., glucose, a fatty acid, an amino acid, glutamine, glycogen, pyruvate), a salt, and/or a mineral. The constituent may be extracted from the media by at least a portion of cells. The constituent may be secreted into the media by at least a portion of the cells.

It should further be appreciated that certain embodiments of the flux block <NUM> of the present disclosure can be formed or made of two or more, such as two separate, detachable parts including: (<NUM>) a first frame; and (<NUM>) a second frame. The first frame and the second frame, respectively, include a first and second attachment mechanism that respectively allows the first frame to be (permanently or removably) attached to the second frame and vice versa.

The present disclosure also provides kits comprising one or more flux blocks sealed in a foil bag. It has been found that flux blocks can be maintain for up to <NUM> days if sealed in foil bags.

This example demonstrates how the flux block of the present disclosure can be used to determine correction factors for a flux analyzer. In this example, five flux blocks having the general arrangement of <FIG> were evaluated to calculate a sensor correction factor from repeated measurements of the sensor in each well in each flux block. As described above, the correction factor is applied in calibration to calibrate the flux analyzer. By applying a correction factor to calibrate the flux analyzer, the coefficient of variance for the data collected from the sensors improves.

The five flux blocks included <NUM> wells. Each flux block was evaluated in four trials, for a total of <NUM> trials. The chamber was filled with <NUM>% CO2 gas, and the wells were loaded with a small volume of phosphate buffered saline. ECAR and OCR data were measured in each well of each flux block using a Seahorse XF24 Analyzer (more particularly, using the fluorescent sensors in that analyzer). The data was further used to calculate a correction factor to be applied for calibrating each sensor of the flux analyzer, as described below.

Data related to calculating the correction factor is listed in Tables <NUM> through 6B; this data is generally representative of testing and use of the flux block of the present disclosure. In those tables, no recharge refers to a series of tests where the gas in the chamber is not replenished between tests and recharge refers to a series of tests where the gas in the chamber is replenished between tests. As shown in Table <NUM>, the first flux block (labeled FB1) was tested four times. ECAR and OCR data were measured in each of the twenty-four wells in each trial. The same data were collected in each trial when evaluating the other four flux blocks.

Upon collecting all ECAR and OCR data for each trial of each flux block, the average ECAR and OCR for each well of all five flux blocks were calculated. This data is represented as "AVG" in Table <NUM>. For example, the average ECAR measurement for well <NUM> in the five flux blocks evaluated was <NUM>. The average ECAR measurement and the average OCR measurement were calculated for each well across the five flux blocks. In this manner, an average sensor measurement can be obtained for a given sensor, by repeated measurements with a single flux block or multiple measurements with several flux blocks.

To calculate a sensor correction factor, the average ECAR and OCR measurements from each sensor (that is, from wells in the same position across all five flux blocks) were calculated. These measurements were calculated by averaging each average ECAR and OCR reading of each well (well <NUM>, well <NUM>,. well <NUM>) of the five flux blocks. Additionally, the standard deviation of ECAR and OCR measurements across all wells of a flux block as calculated from each ECAR and OCR average reading for each well of the five flux blocks. The same sensor was used in the same well of all five flux blocks; for example, sensor <NUM> of the analyzer was used to measure ECAR and OCR in well <NUM> in each of the five flux blocks. The average ECAR and OCR and the standard deviation for ECAR and OCR measurements were used to calculate the coefficient of variation of ECAR and OCR for any flux block. As seen in Table <NUM>, the coefficient of variation for ECAR measurements was <NUM>% and the CV for OCR measurements was <NUM>%. This data shows good interwell reproducibility of ECAR and OCR measurements amongst the five flux blocks.

Furthermore, a correction factor for each well of a flux block was calculated by dividing the average ECAR measurement of a flux block by the average ECAR among the measurements from each sensor in the analyzer. As seen in Table 7A, the average coefficient of variation was calculated from the coefficients of variation of each of the flux blocks. This average CV was <NUM>. Furthermore, Table 7B shows how the CV decreases, and therefore the reproducibility of results improves, when applying the correction factor to data collected from using the flux block. In Table 7B the correction factor was used as a multiplier by the average ECAR measurement from each well of a flux block to equate to a calibrated average ECAR measurement from each well of a flux block. The average ECAR measurement of each well of a flux block was adjusted in this manner. Furthermore, the corrected average ECAR measurement of each well of a flux block was averaged to equate to an average coefficient of variation of <NUM>%. Thus, by applying the correction factor, the average coefficient of variation of the <NUM>-sensor analyzer was lowered by <NUM>%, thus showing that applying the correction factor when calibrating the flux analyzer provides improved results.

This example compares a flux block of the present disclosure to a biological (cell-based) standard in taking measurements from a flux analyzer. In this example, the flux blocks had the general arrangement of <FIG>, though some flux blocks had <NUM> wells. A flux block and a cell-based standard were each used to obtain OCR and ECAR measurements from Seahorse XF24 and XF96 flux analyzers. The cell-based standard was a confluent monolayer of cells (C2C12 skeletal mouse fibroblasts). Table 8A shows data from the XF24 analyzers, and Table 8B shows data from the XF96 analyzers; this data is generally representative of testing and use of the flux block of the present disclosure. The tables report the coefficients of variance (CV) among wells of the standards (which is the CV for the sensors of the analyzer used with those wells). The rates/flux of OCR and ECAR measured by each analyzer (calculated from all <NUM> or <NUM> sensors of the analyzer) are also next. For OCR the CV between the different well plates is calculated, and is <NUM>% when the cell-based standards were used but only <NUM>% when the flux blocks were used. For ECAR, the CV was <NUM>% for cell-based standards and <NUM>% for the flux blocks. Thus, the flux blocks produced measurements having far less variation due to the standard (as opposed to variation attributable to the analyzer) than the cell-based standards.

With regard to interwell variance (that is, the CV from the <NUM> or <NUM> measurements of the individual wells of a single well-plate), some of the measurements using the flux blocks resulted in higher CV numbers than the cell-based standards. For the XF24 analyzers, the CV% of ECAR was <NUM>% for the cell-based assay and <NUM>% for the flux blocks. For the XF96 analyzers, the CV% of OCR was <NUM>% for the cell-based standard and <NUM>% for the flux blocks. However, at least some of this variance is attributed to the sensors rather than the standards.

The present invention relates to a apparatus as defined in claim <NUM>, a method of calibrating a flux analyzer using said apparatus as defined in claim <NUM> and a method of making such apparatus as defined in claim <NUM>. Preferred embodiments of the claimed apparatus and the claimed methods are defined in the dependent claims. The features of the preferred embodiments can be arbitrarily combined with each other. Any "embodiment" or "example" which is disclosed in the description but not covered by the claims should be considered as presented for illustrative purpose only.

Claim 1:
A device comprising a flux analyser and an apparatus for calibrating said flux analyser, wherein said flux analyser measures the change of O2, CO2, or both as a function of time,
wherein said apparatus for calibrating the flux analyzer comprises:
a first frame (<NUM>, <NUM>) comprising a plurality of wells (<NUM>, <NUM>), each well having a well opening (<NUM>) at its bottom;
a second frame (<NUM>) connected or integrally formed with the first frame (<NUM>, <NUM>) wherein the second frame (<NUM>) defines a chamber (<NUM>, <NUM>) and an inlet port (<NUM>) to the chamber (<NUM>, <NUM>), and the second frame (<NUM>) has at least one chamber opening (<NUM>), wherein the chamber opening (<NUM>) at least partially overlaps the well openings (<NUM>); and
a selectively permeable membrane (<NUM>, <NUM>) between the first (<NUM>, <NUM>) and second frames (<NUM>) that separates the well openings (<NUM>) from the chamber opening (<NUM>), wherein the selectively permeable membrane (<NUM>, <NUM>) is permeable to O2, CO2, or both, and impermeable to water.