Microtiter plate cap for eliminating headspace and method for optically measuring hole oxygen concentration through cap

Abstract

An appliance for eliminating headspace in one or more test spaces (T9 or MP wells) of a test tube (T) or microtiter plate (MP) and a method of measuring oxygen concentration in a test sample using such an appliance. The appliance protrudes into a test chamber (T9 or MP hole) to displace a portion of a fluid sample within the test chamber (T9 or MP hole), and has longitudinally extending slots (109 and 229) through which the displaced fluid content can be expelled from the test chamber (T9 or MP hole).

Description

This application is a divisional application of Chinese patent application No. 201880032391.1 (filed on May 15, 2018, entitled "Micro-titer plate cover for eliminating headspace and method for optically measuring oxygen concentration in the orifice through the cover"). Background Technology

[0001] Test tubes and microtiter plates are typically used with oxygen-sensitive photoluminescent probes to measure and monitor the aerobic activity of a sample by measuring and monitoring the oxygen concentration within the tube or well. This requires sealing the sample to prevent fluid communication with the surrounding environment, which is typically achieved by providing an oil layer over the sample and probing the oxygen-sensitive photoluminescent probe through the oil layer into the sample. Sealing the sample with an oil layer provides the additional benefit of limiting the gaseous headspace between the sample and the oil layer. Such a gaseous headspace is known to slow the detection of changes in oxygen concentration due to the relatively abundant supply of oxygen available in the gaseous headspace trapped beneath the oxygen barrier layer.

[0002] While sealing the sample to prevent direct fluid communication with the surrounding environment and limiting the presence of gaseous headspace beneath the oil layer is generally effective, the oil layer is not a particularly effective oxygen barrier, is difficult to function correctly and consistently, and is labor-intensive.

[0003] Therefore, there is a substantial need for an effective, rapid, and easy apparatus and technique for effectively sealing a sample in the orifice of a test tube or microtiter plate so as to prevent fluid communication with the surrounding environment, such apparatus and technique not leaving a gaseous headspace between the oxygen barrier layer and the sample and not interfering with the probing of an oxygen-sensitive photoluminescent probe in the sample through the apparatus. Summary of the Invention

[0004] An apparatus for eliminating headspace in a test chamber, such as a stopper for a test tube or a cap for a porous microtiter plate, and a method for using such an apparatus in combination with one or more test chambers to measure oxygen concentration.

[0005] One embodiment of the apparatus is a plug formed of an oxygen-barrier material, the plug being configured and arranged to project longitudinally and sealingly into the cavity of a test tube, the plug having an outwardly projecting convex distal end and a plurality of longitudinally extending grooves operable to provide a peripheral outlet channel between the plug and the test tube, through which fluid contents displaced within the cavity of the test tube by inserting the plug into the cavity of the test tube can be discharged from the cavity.

[0006] Another embodiment of the apparatus is a cap made of an oxygen-barrier material for a microtiter plate having any array of pores. The cap includes (A) a cover plate for engaging the microtiter plate, and (B) protrusions extending longitudinally from the cover plate in an array corresponding to the array of pores, wherein each protrusion (i) is configured and arranged longitudinally and sealably protrudes into a corresponding pore of the microtiter plate, (ii) has an outwardly projecting convex distal end, and (iii) has a plurality of longitudinally extending grooves operable to provide a peripheral outlet channel between the protrusion and the pore, through which fluid contents displaced within the pore by insertion of the protrusion into the pore can be discharged from the pore.

[0007] The cap can be combined with a microtiter plate formed of an oxygen-barrier material and having a pore array to form an assembly. The cap is configured and arranged to engage with the microtiter plate, wherein the protrusions extend longitudinally from the cap in an array corresponding to the pore array of the microtiter plate, such that when the cap is placed on the microtiter plate, the protrusions extend into the pores.

[0008] The oxygen concentration in a test tube can be measured using a plug embodiment of the apparatus by: (A) placing an oxygen-sensitive photoluminescent material and a fluid test sample into the cavity of the test tube; (B) frictionally inserting the apparatus into the cavity of the tube to form a sealed chamber, forming a peripheral outlet channel between the apparatus and the test tube, through which fluid contents displaced within the cavity of the test tube by inserting the apparatus into the cavity can be discharged from the cavity through the peripheral outlet channel; and (C) determining the oxygen concentration in the sealed chamber by: (i) exposing the oxygen-sensitive photoluminescent material in the sealed chamber to excitation radiation passing through the apparatus to generate excited oxygen-sensitive photoluminescent material; (ii) measuring the radiation emitted by the excited oxygen-sensitive photoluminescent material through the apparatus; and (iii) converting the measured emission into a target analyte concentration based on a known conversion algorithm.

[0009] The oxygen concentration in an array of wells of a microtiter plate can be measured using the plug embodiment of the apparatus by: (A) placing an oxygen-sensitive photoluminescent material and a fluid test sample into a plurality of wells of the microtiter plate; (B) covering the microtiter plate with the cover plate, whereby each protrusion extends into and seals into each well of the microtiter plate, thereby displacing fluid from within each well to the periphery of the protrusion and out of the well through a peripheral outlet channel formed between the protrusion and the well; and (C) determining the oxygen concentration in each well of the covered microtiter plate by: (i) exposing the oxygen-sensitive photoluminescent material in each well to excitation radiation through the protrusion extending into the well to generate excited oxygen-sensitive photoluminescent material; (ii) measuring the radiation emitted by the excited oxygen-sensitive photoluminescent material through the protrusion; and (iii) converting the measured emission into a target analyte concentration based on a known conversion algorithm. Attached Figure Description

[0010] Figure 1 This is a side view of the first embodiment of the invention above a standard test tube.

[0011] Figure 2 It is an insertion. Figure 1 The test tube shown Figure 1 An enlarged top view of the present invention is shown.

[0012] Figure 3 This is a perspective view of a second embodiment of the present invention, which has a single row of protrusions array.

[0013] Figure 4 yes Figure 3 Another perspective view of the lid shown.

[0014] Figure 5 yes Figure 3 The top view of the cover is shown.

[0015] Figure 6 yes Figure 3 The bottom view of the lid is shown.

[0016] Figure 7 yes Figure 3 The right-side view of the lid is shown.

[0017] Figure 8 yes Figure 3 The left-side view of the lid is shown.

[0018] Figure 9 It was taken from BB along the line. Figure 5 The illustrated cross-sectional view of the cover.

[0019] Figure 10 It is a cut along the CC line. Figure 5 The illustrated cross-sectional view of the cover.

[0020] Figure 11 yes Figure 3 The end view of the lid is shown.

[0021] Figure 12 This is a perspective view of a third embodiment of the invention suitable for use as a cap for a standard 96-well microtiter plate.

[0022] Figure 13 This is a top view of a standard 96-well microtiter plate. Detailed Implementation Naming Table

[0023] The present invention is a stopper for use in the cavity T9 of a test tube T or the orifice MP of a microtiter plate MP. 孔 The fluid (mainly gaseous headspace) is displaced without affecting the flow of light through the central optical path to the cavity T9 of test tube T or the orifice MP of microtiter plate MP. 孔 Top-down exploration of oxygen-sensitive photoluminescent materials.

[0024] Reference Figure 1 and Figure 2 A first embodiment of the invention is a plug 100, which is configured and arranged for use with a separate test tube T. The plug 100 has a proximal end 101 and a convex distal end 102, wherein a series of grooves 109 extend along the longitudinal length x of the plug 100. When inserted into the cavity T9 of the test tube T, the grooves 109 engage with the inner wall (unnumbered) of the test tube T to form a peripheral outlet channel 119 through which fluid displaced from the cavity T9 of the test tube T upon insertion of the plug 100 may exit the cavity T9.

[0025] The plug 100 preferably has (-) a longitudinal length between 0.5 cm and 2 cm, (-) a convex distal end 102, the radius of curvature of which is between about 2 and 10 times the outer width y or z of the plug 100, and (-) longitudinal x-extending grooves 109 that are evenly spaced on the circumference between 2 and 10, more preferably between 2 and 6, and most preferably between 4 and 6.

[0026] When the plug 100 is sealed within the cavity T9 of the test tube T, the groove 109 is preferably configured, arranged, and sized, and formed with a diameter of 0.2 mm. 2 With 4mm 2 The outer outlet channel 119 of the radial section between.

[0027] The plug 100 is preferably made of an oxygen barrier material, and most preferably has a thickness of less than 16 cm at 23°C and 0% RH. 3 / m 2 The material is formed with an oxygen permeability of / 24h.

[0028] Reference Figure 3-12 A second embodiment of the invention is a cover 200, which includes a cover plate 210 for engaging a microtiter plate MP, and a protrusion array 220 extending longitudinally x from the cover plate 210 to engage with the orifice MP of the microtiter plate MP. 孔 They match and mate. Each protrusion 220 has a proximal end 221 and a convex distal end 222, wherein a series of grooves 229 extend along the longitudinal length x of the protrusion 220. When the cover plate 210 is attached to the microtiter plate MP, each protrusion 220 extends into an orifice MP on the microtiter plate MP. 孔 In the middle. When inserted into the hole MP 孔 In the middle, the groove 229 on each protrusion 220 corresponds to the hole MP. 孔 The inner wall (unnumbered) fits to form a peripheral outlet channel, which opens from the hole MP when the protrusion 220 is inserted. 孔 The displaced fluid can exit the orifice MP through the peripheral outlet channel. 孔 .

[0029] Each cap 200 preferably has a uniform array of 6, 24, 96, 384, or 1536 protrusions, the array being configured, arranged, and sized to have the same number of holes MP as the microtiter plate MP. 孔 pair.

[0030] Each protrusion 220 preferably has (-) a longitudinal x-length between 4 mm and 12 mm, (-) a convex distal end 222, the radius of curvature of which is between approximately 2 and 10 times the outer width y or z of the protrusion 220, and (-) longitudinal x-extending grooves 229 evenly spaced circumferentially between 2 and 10, more preferably between 2 and 6, and most preferably between 4 and 6. All protrusions 220 on the cover 200 are preferably of uniform size.

[0031] When protrusion 220 sealably engages with the orifice MP on the microtiter plate MP 孔 When internal, the groove 229 is preferably configured, arranged, and sized, and forms a groove with a diameter of 0.1 mm. 2 With 0.4mm 2 The peripheral outlet channel of the radial section between.

[0032] The peripheral outlet channel is preferably in fluid communication with the atmosphere through an opening (not numbered) in the cover plate 210.

[0033] The cover 200 is preferably made of an oxygen-barrier material, and most preferably has a thickness of less than 16 cm at 23°C and 0% RH. 3 / m 2 The material with an oxygen permeability of 24h is formed into a single piece.

[0034] Oxygen-sensitive photoluminescent probes capable of sensing and reporting the oxygen concentration in an environment in fluid communication with the probe are well known. See, for example, U.S. Patent Application Publications 2011 / 0136247, 2009 / 0029402, 2008 / 199360, 2008 / 190172, 2007 / 0042412, and 2004 / 0033575; U.S. Patents 8,242,162, 8,158,438, 7,862,770, 7,849,729, 7,749,768, 7,679,745, 7,674,626, 7,569,395, 7,534,615, 7,368,153, and 7,130. 8,270, 6,989,246, 6,689,438, 6,395,506, 6,379,969, 6,080,574, 5,885,843, 5,863,460, 5,718,842, 5,595,708, 5,567,598, 5,462,879, 5,407,892, 5,114,676, 5,094,959, 5,030,420, 4,965,087, 4,810,655, and 4,476,870; PCT International Publication WO 2008 / 146087; and European Publication Patent Application EP 1134583, all of which are incorporated herein by reference. Such optical sensors are available from a number of suppliers, including Presens Precision Sensing, GmbH in Regensburg, Germany; Oxysense in Dallas, Texas, USA; and Luxcel Biosciences, Ltd in Cork, Ireland.

[0035] Methods and techniques for sensing oxygen in the wells of test tubes or microtiter plates using oxygen-sensitive photoluminescent probes are well known, as illustrated in WO 2012 / 052068, U.S. Patent Application Publication 2013 / 0280751, and U.S. Patent Application Publication 2014 / 0147882, all of which are incorporated herein by reference. These methods and techniques are applicable to determining the oxygen concentration in test tubes or wells sealed with the apparatus according to the invention.

[0036] Instruments suitable for reading oxygen-sensitive photoluminescent probes in the wells of microtiter plates are known and available from many sources, including the CLARIOstar plate reader from BMG Labtech GmbH in Altenberg, Germany.

[0037] The oxygen concentration in a test tube T can be measured and monitored using the plug 100 according to a first embodiment of the invention by: (a) placing an oxygen-sensitive photoluminescent material and a fluid test sample into a cavity T9 of the test tube T; (b) inserting the plug 100 into the cavity T9 of the test tube T in a frictional engagement manner to form a sealed chamber, wherein a peripheral outlet channel 119 is formed between the plug 100 and the test tube T, through which fluid contents in the cavity T9 of the test tube T displaced by inserting the plug 100 into the cavity T9 of the test tube T can be discharged from the cavity T9 through the peripheral outlet channel; and (c) determining the oxygen concentration in the sealed chamber by: exposing the oxygen-sensitive photoluminescent material in the sealed chamber to excitation radiation passing through the plug 100 to generate excited oxygen-sensitive photoluminescent material, measuring the radiation emitted by the excited oxygen-sensitive photoluminescent material passing through the plug 100, and converting the measured emission into a target analyte concentration based on a known conversion algorithm.

[0038] Similarly, the cap 200 according to the second embodiment of the invention can be used to measure and monitor each well MP of a microtiter plate. 孔 Oxygen concentration within: (a) placing oxygen-sensitive photoluminescent materials and fluid test samples into multiple wells of a microtiter plate MP. 孔 In each of the following, (b) the microtiter plate MP is covered with a cover 200 according to a second embodiment of the invention, whereby when the cover 210 is placed and secured to the microtiter plate MP, each protrusion 220 on the cover 200 extends into and sealably engages with each hole MP of the microtiter plate MP. 孔 Inside, so that fluid flows from each orifice MP 孔 The outer periphery of the inward protrusion 220 is shifted and through the protrusion 220 and the hole MP 孔 The outer exit channel formed between them is from the hole MP 孔 Remove from the center, and (c) determine each well MP of the covered microtiter plate MP by means of the following manner. 孔 Oxygen concentration inside: MP per well 孔 An oxygen-sensitive photoluminescent material is exposed to excitation radiation passing through a protrusion 220 extending into the hole to generate an excited oxygen-sensitive photoluminescent material. The radiation emitted by the excited oxygen-sensitive photoluminescent material through the protrusion 220 is measured, and the measured emission is converted into the concentration of the target analyte based on a known conversion algorithm. This disclosure relates to the following implementation plans. 1. An apparatus comprising a plug formed of an oxygen-barrier material, the plug being configured and arranged to project longitudinally and hermetically into a cavity of a test tube, the plug having an outwardly projecting convex distal end and a plurality of longitudinally extending grooves operable to provide a peripheral outlet channel between the plug and the test tube, through which fluid contents displaced within the cavity of the test tube by insertion of the plug into the cavity of the test tube may be discharged from the cavity. 2. The appliance according to claim 1, wherein the plug has a longitudinal length and a lateral external width, and The radius of curvature of the convex distal end of the plug is approximately 2 to 10 times the outer width of the plug. 3. The appliance according to any one of claims 1 or 2, wherein the plug is made of material having a temperature of 23°C and 0% RH. Less than 16cm 3 / m 2 The material is formed with an oxygen permeability of / 24h. 4. The appliance according to any one of items 1-3, wherein the plug has a longitudinal length between 0.5 cm and 2 cm. 5. The appliance according to any one of items 1-4, wherein the plug has longitudinally extending grooves between 2 and 10. 6. The appliance according to any one of items 1-4, wherein the plug has longitudinally extending grooves between 2 and 6. 7. The appliance according to any one of items 1-4, wherein the plug has longitudinally extending grooves evenly spaced circumferentially between 4 and 6. 8. The apparatus according to any one of claims 1-7, wherein when the plug is sealed into the cavity of the test tube, the groove is formed having a depth of 0.2 mm. 2 With 4mm 2 The peripheral outlet channel of the radial section between. 9. A cap for a microtiter plate having any array of pores, the cap being formed of an oxygen barrier material. It includes: (a) A cover plate for engaging the microtiter plate, and (b) Protrusions extending longitudinally from the cover plate in an array corresponding to the array of orifices, wherein each protrusion (i) is configured and arranged longitudinally and sealingly projecting into a corresponding orifice of the microtiter plate, (ii) has an outwardly projecting convex distal end, and (iii) has a plurality of longitudinally extending grooves operable to provide a peripheral outlet channel between the protrusion and the orifice, displaced by inserting the protrusion into the orifice. The fluid contents inside the orifice can be discharged from the orifice through the peripheral outlet channel. 10. The cover according to claim 9, wherein the protrusion has a longitudinal length and a lateral outward width, and The radius of curvature of the convex distal end of each protrusion is between approximately 2 and 10 times the outer width of the protrusion. 11. The cover according to any one of claims 9 or 10, wherein the cover is made of material having a thickness of less than 16 cm at 23°C and 0% RH. 3 / m 2 The material is formed with an oxygen permeability of / 24h. 12. The cover according to any one of items 9-11, wherein the cover is a single piece. 13. The cover according to any one of items 9-12, wherein each protrusion has a longitudinal length between 4 mm and 12 mm. 14. The cover according to item 13, wherein the protrusion has the same longitudinal length between 6 mm and 12 mm. 15. The cover according to any one of items 9-14, wherein each protrusion has a longitudinally extending groove between 2 and 10. 16. The cover according to any one of items 9-14, wherein each protrusion has a longitudinally extending groove between 2 and 6. 17. The cover according to any one of items 9-14, wherein each protrusion has longitudinally extending grooves that are evenly spaced circumferentially between four and six. 18. The cover according to any one of claims 9-17, wherein the peripheral outlet channel is in fluid communication with the atmosphere through an opening in the cover plate. 19. The cover according to any one of claims 9-18, wherein when the protrusions are sealingly engaged within the hole, the grooves on each protrusion are formed having a depth of 0.1 mm. 2 With 0.4mm 2 The peripheral outlet channel of the radial section between. 20. The cover according to any one of claims 9-19, wherein the cover has a uniform array of 6, 24, 96, 384 or 1536 protrusions. 21. A component comprising: (a) A microtiter plate formed of an oxygen-barrier material and having a pore array, and (b) The cap according to any one of items 9-19, the cap being configured and arranged to engage with the microtiter plate, wherein the protrusion extends longitudinally from the cap in an array corresponding to the array of holes of the microtiter plate, such that when the cap is placed on the microtiter plate, the protrusion extends into the holes. 22. A method for measuring the oxygen concentration in a test tube, comprising the following steps: (a) Place the oxygen-sensitive photoluminescent material and the fluid test sample into the cavity of the test tube. (b) The instrument according to any one of items 1-8 is frictionally inserted into the cavity of the tube to form a sealed chamber, and a peripheral outlet channel is formed between the instrument and the test tube, through which fluid contents displaced within the cavity of the test tube by the insertion of the instrument into the cavity of the test tube may be discharged from the cavity through the peripheral outlet channel, and (c) The oxygen concentration in the sealed chamber is determined by: (i) exposing the oxygen-sensitive photoluminescent material in the sealed chamber to excitation radiation passing through the apparatus to generate excited oxygen-sensitive photoluminescent material; (ii) measuring the radiation emitted by the excited oxygen-sensitive photoluminescent material passing through the apparatus; and (iii) based on A known conversion algorithm converts measured emission into target analyte concentration. 23. The method according to item 22, wherein the device is hollow and has a closed distal end and an open proximal end. 24. The method according to any one of items 22 or 23, wherein the distance of insertion of said instrument is sufficient to displace the fluid test sample into each peripheral outlet channel. 25. A method for measuring the oxygen concentration within a well array of a microtiter plate, comprising the following steps: (a) Obtain the component as described in item 21 (b) Place the oxygen-sensitive photoluminescent material and the fluid test sample into multiple wells of the microtiter plate. (c) The microtiter plate is covered with the cover plate, whereby each protrusion extends into and seals into each orifice of the microtiter plate, thereby displacing fluid from within each orifice to the periphery of the protrusion and out of the orifice through a peripheral outlet channel formed between the protrusion and the orifice, and (d) Determine the oxygen concentration in each well of the covered microtiter plate by: (i) Exposing the oxygen-sensitive photoluminescent material within each pore to excitation radiation passing through protrusions extending into the pore to generate excited oxygen-sensitive photoluminescent material, (ii) (iii) Measure the radiation emitted by the excited oxygen-sensitive photoluminescent material through the protrusion, and (iii) convert the measured emission into target analysis based on a known conversion algorithm. Concentration of substance. 26. The method according to item 25, wherein each protrusion is hollow, having a closed distal end and an open proximal end. 27. The method according to any one of items 25 or 26, wherein a sufficient amount of fluid test sample is placed in each well such that when the cover plate is fixed to the microtiter plate, the fluid test sample is displaced into each peripheral outlet channel of each well. 28. The method according to any one of claims 22-27, wherein the oxygen-sensitive photoluminescent material comprises an oxygen-sensitive indicator dye incorporated into an oxygen-permeable polymer matrix. 29. The method according to any one of claims 22-28, wherein the oxygen-sensitive photoluminescent material comprises nanoparticles containing an oxygen-sensitive indicator dye. 30. The method according to any one of items 22-29, wherein the fluid test sample contains live cells. 31. The method according to claim 30, wherein the living cell is a living mammalian cell. 32. The method according to any one of items 22-29, wherein the fluid test sample contains live microorganisms.

Claims

1. An implement comprising a plug formed of an oxygen barrier material, the plug being configured and arranged to project longitudinally and sealingly into a cavity of a test tube, the plug having an outwardly projecting convex distal end and a plurality of longitudinally extending slots operable to provide a peripheral exit passageway between the plug and the test tube through which fluid contents within the cavity of the test tube displaced by insertion of the plug into the cavity of the test tube can be expelled from the cavity.

2. The implement of claim 1, wherein the plug has a longitudinal length and a lateral outer width, and the radius of curvature of the convex distal end of the plug is between about 2 and 10 times the outer width of the plug.

3. An appliance according to either one of claims 1 or 2, wherein the plug is formed from a material having an oxygen transmission rate of less than 16 cm3 3 / m 2 / 24h at 23 °C and 0% RH.

4. The implement of any one of claims 1-3, wherein the plug has a longitudinal length of between 0.5 cm and 2 cm.

5. The implement of any one of claims 1-4, wherein the plug has between 2 and 10 longitudinally extending slots.

6. The implement of any one of claims 1-4, wherein the plug has between 2 and 6 longitudinally extending slots.

7. The implement of any one of claims 1-4, wherein the plug has between 4 and 6 longitudinally extending slots evenly spaced circumferentially.

8. The apparatus of any one of claims 1-7, wherein when the plug sealingly engages within the lumen of the test tube, the slot forms a peripheral outlet passageway having a radial cross-section between 0.2 mm 2 and 4 mm 2 .

9. A cover for a microtiter plate having any array of wells, the cover being formed of an oxygen barrier material and comprising: (a) a cover plate for engaging the microtiter plate, and (b) protrusions extending longitudinally from the cover plate in an array corresponding to the array of wells, wherein each protrusion (i) is configured and arranged to project longitudinally and sealingly into a corresponding well of the microtiter plate, (ii) has an outwardly projecting convex distal end, and (iii) has a plurality of longitudinally extending slots operable to provide a peripheral exit passageway between the protrusion and the well through which fluid contents within the well displaced by insertion of the protrusion into the well can be expelled from the well.

10. The cover of claim 9, wherein the protrusions have a longitudinal length and a lateral outer width, and the radius of curvature of the convex distal end of each protrusion is between about 2 and 10 times the outer width of the protrusion.

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