CA2348564A1 - Ultrathin-walled multiwell plate for heat block thermocycling - Google Patents
Ultrathin-walled multiwell plate for heat block thermocycling Download PDFInfo
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- CA2348564A1 CA2348564A1 CA002348564A CA2348564A CA2348564A1 CA 2348564 A1 CA2348564 A1 CA 2348564A1 CA 002348564 A CA002348564 A CA 002348564A CA 2348564 A CA2348564 A CA 2348564A CA 2348564 A1 CA2348564 A1 CA 2348564A1
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- wells
- ultrathin
- heat block
- multiwell plate
- walls
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
- B01L3/50851—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates specially adapted for heating or cooling samples
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L7/00—Heating or cooling apparatus; Heat insulating devices
- B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
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Abstract
Ultrathin-walled multiwell reactors for heat block thermocycling of samples comprising an array of small-volume wells of identical height with similarly shaped sample wells formed in the top surface of the heat block of the thermocycler are provided. The multiwell plates are preferentially vacuumformed out of a 30-50 micron thick thermoplastic film and can be used for rapid, oil-free temperature cycling of small (1-10.mu.l) volume samples.
Description
Ultrathin-walled multiwell plate for heat block thermocycling The invention relates to plastic plates for conventional heat block thermocycling s of biological samples, particularly to rnultiweil plates. More specifically, it relates to ultrathin-walled multiwell plates with an improved heat transfer to small-volume samples. Such plates can be used for rapid temperature cycling of multiple, small-volume samples (i.e. 1-20 pl) by using heat block thermocyclers with an increased block temperature ramping rate (i.e. 4 ° C/second and greater) and standard heated-lid ~ o technology for sealing the samples.
Temperature cycling of biological samples is a central moment in DNA
amplification by the palymerase chain reaction (PCR:) (Saiki et al., Science, 239, 487-491 [1988]). Much effort is being expended in developing various alternative reactors and technologies for rapid temperature cycling of small-volume samples (Kopp et al., Science ~5 280, 1046-1048 [1998]; Belgrader et al., ].Forensic Science 43, 3i5-319 [1998]; Wittwer et al., Analytical Biochem., 186, 328-331 [1990] and U.S. Patent No 5,4SS;17S;
Woolley et al., Analytical Chem., 68, 4081-4086 [(i996]).
One commercially available type of microreactor and thermocycler for rapid temperature cycling of small samples is a glass capillary tube and a hot-air thermocycler zo from Roche Molecular Biochemicals (cat No. 1909 ?.39 and cat No. 20I 1468, respectively).
The glass capillary tube can hold reaction volumes ranging from 10 to 20 p.l.
The hot-air thermocycler can hold 32 capillaries and perform 30 - 40 PCR cycles in 20-30 minutes.
However, these rapid DNA amplification technology is connected with various disadvantages, for example:
25 a) The handling of the individual capillaries is relatively cumbersome.
b) The relatively large glass surface adsorbs components of the standard PCR-mixtures.
This might inactivate the reaction. Therefore, various carrier molecules, i.e.
proteins or even DNA, must be added and the concentrations of the components reoptimized.
c) The cost of the capillary tube, as a disposable PCR container, is high when compared 3o to the standard 0.2 ml PCR tube.
d) The experimental throughput using this system is limited.
It is surprising that only little research has been conducted to improve the basic performance in sample size and speed of the widely used, conventional heat block thermocycling of samples contained in plastic tubes or multiwell plates. One known improvement of heat block temperature cycling of samples contained in plastic tubes has been described by Half et al. (Biotechniques, 10, lOEi-112, j1991] and U.S.
Patent No 5,475,610). They describe a special PCR reaction-compatible one-piece plastic s microcentrifuge tube, i.e. a thin-walled PCR tube. The tube has a cylindrically shaped upper wall section, a relatively thin (i.e. approximately 0.3 rnm) conically-shaped lower wall section and a dome-shaped bottom. The samples as small as 20 p,1 are placed into the tubes, the tubes are closed by deformable, gas-tight caps and positioned into similarly shaped conical wells machined in the body of the heat block. The heated cover ~o compresses each cap and forces each tube down firmly into its own well. The heated platen (i:e. heated lid) serves several goals by supplying the appropriate pressure to the caps of the tubes: it maintains the conically shaped walls in close thermal contact with the body of the block; it prevents the opening of the caps. by increased air pressure arising in the tubes at elevated temperatures. In addition, it maintains the parts of the tubes that ~s project above the top surface of the block at 95° -100° C in order to prevent water condensation and sample loss in the course of thermocycling. This made it possible to exclude the placing of mineral oil or glycerol into the; wells of the block in order to improve the heat transfer to the tubes and the overlaying of the samples by mineral oil that prevented evaporation but also served as added tlhermal mass. In addition, the PCR
zo tubes can be put in a two-piece holder (US patent 5,710,381) of an 8x12, 96-well microplate format, which can be used to support the high sample throughput needs with any number between 1 and 96 individual reaction tubes.
In DE 4022792 the inventors describe a plate with cylindrically shaped walls of the wells and spherically shaped bottoms thereof. ThE; individual wells of the plate were zs formed by melting a polycarbonate sheet in the range of 0.27-0.5 mm by a stream of hot air. This technology leads to relatively thin walls in the range of 0.08-0.2mm. The biological samples were placed into the wells, covered with polycarbonate film (0. i mm) and the individual wells were thermosealed by a special press. Upon sealing the plate was placed on the thermoblock and fixed by screws. Though theoretically the heat transfer to 3o the samples is improved, however, the way of positioning the plate on the block and the cylindrical and spherical geometry of the well prevent a close thermal contact with the heating block. During thermocyling, due to the large thermal expansion, the plate fixed by WO OOI25920 PCTl~P99/0817g screws becomes deformed and the close thermal contact is not maintained anymore.
Therefore, by using the above technology rapid cycling reactions cannot be performed.
The other known improvement of heat block thermocycling is described in PCT
patent application WO 98/43740. It concerns a neat block thenmocycler with an increased ramping rate, i.e. 4° Clsecond). The thermocycler ca~;~ hold 96 PCR
tubes (each of a volume of 0.2 ml) or 96-well PCR plates. Theoretica,lIy, the thermocycler can perform 30 PCR cycles in 20-30 minutes, provided that only a fe;w seconds are spent to reach the temperature equilibrium between the heat block and the samples.
However, as described in U.S. Patent No 5,508,197, even if the temperature of the ~o heat-transfer media; i.e. water, is changed almost instantaneously, it takes approximately 15 seconds to reach equilibrium between water and t:he 15-20 p.l samples in the standard PCR plates. This means that for 30 PCR cycles approximately 20 minutes are spent to reach the equilibrium between heat-transfer media ar.~d the 15-20 ul samples in the plates.
In comparison, the above mentioned heat block cycler (WD 98/43740) operating ~s at a ramping rate of 4° C/second, needs for the heat-block temperature transitions during 30 PCR cycles 10 minutes only. This shows that the :major limiting factor for rapid temperature cycling of small samples in platic PCR tubes or PCR plates is the low efficiency of the heat transfer through the walls of conventional PCR tubes or plates, respectively.
zo The present invention concerns plastic multiwell plates for performing heat block thermocycling of multiple samples. More specifically, it concerns ultrathin-walled multiwell plates with an improved heat transfer to small samples. Ultrathin-walled multiwell plates are suited for rapid, oil-free, heat block temperature cycling of small-volume samples (i.e. approximately 1-20 p,l), wherea:> the Lower limit is given by the zs reliability of the conventional pipetting systems.
Figure 1 illustrates an example of a multiwell plate according to the invention.
Figure 2 illustrates the positioning of the plate in the )block of the thermal cycler.
One aspect of the present invention concerns the considerably decreased thickness (i.e. approximately 7.5-15 fold) of the well walls when compared to known thin-walled 3o PCR tubes (U.S. Patent No 5,475,610.). This can be reached, for example, by means of thermoforming the plates out of thin thermoplastic films. Such thermoplastic films are, for example, polyolefin films, such as metallocene-catalyzed polyolefin films and/or copolymer films. Usually, the multiwell plate is vacuumformed out of cast, unoriented polypropylene fzlm, polypropylene-polyethylene copolymer films or metallocene-catalyzed polypropylene films. The film is formed into a negative ("female") mould comprising a plurality of spaced-apart, sonically shaped wells which are machined in the body of a mould in the shape of rectangular- or square-array. The thickness of the film s for vacuumforming sonically shaped wells is chosen according to the standard rule used for thermoforming, i.e. thickness of the film = well draw ratio x thickness of the wall of the formed well.
For example, vacuumforming wells with a draw ratio of two and an average thickness of the walls of 30 microns results-in a film thickness of 60 microns. The ~o average optimum wall thickness was found to be 20-~40 microns. The thickness of the well is reduced 7.5-15 fold when compared to the wall thickness of the formerly improved PCR tube desribed in U.S. Patent No 5,475,610. Using the Fourier equation for heat transfer and the equation for temperature transfer through solid substances, it can be shown that heat transfer through one square millimeter of the surface of the well of the ~s plate is increased 7.5-15 fold and the time of temperature transfer through the wall is decreased 56-225 fold when compared to the said PC'R tube. This drastic decrease in time can be explained by the fact that the time needed for ahe transfer of temperature front is proportional to the square power of distance. It can be easily calculated that the time of the temperature transfer through the ultrathin walls of the mufti-well plate is in the range Zo of milliseconds, whereas for the said PCR tube (LT.S. Patent No 5,475,610) it is in the range of seconds. This explains the well known fact that thin (20-40 microns) plastic films are poor thermo insulators.
The thickness of the walls of the formed well:. is gradually reduced to the bottoms of the wells due to vacuumforming of the wells into a~ negative mould. This geometry of zs the walls of the wells provides several advantages:
~ The relatively thick upper parts of the walls of the wells cause additional rigidity of the whole multiwell plate.
~ During heating of the heat block of the thermocycl.er, a vertical temperature gradient is formed in the sample, due to the gradient of the wf;ll-wall thickness. This vertical 3o temperature gradient causes intensive convective mixing of the sample in sonically shaped wells and increases the heat transfer through the sample. In comparison, this convective mixing of the sample is much less eff dent in conventional PCR
plates/tubes with a uniform wall thickness.
Another aspect of the invention concerns the height of the wells of the rnultiwell plate. The height of the conically shaped wells is equal to the height of the similarly shaped sample wells machined in the body of the heat block. Thus, this geometry of the wells (2) enables the positioning of the plate (1) on i:he heat block (4) as shown in Figure s 2. As shown (Figure 2), in contrast to the conventional PCR plates, the walls of the wells (2) of the mufti-well plate (1) do not project above tile top surface of the block {4). The type of positioning provides several advantages: Thc; pressure caused by the screw (12) to the lid (10) {heating element (11)) can be increased i.n order to obtain efficient sealing of the samples (9) sealed, for example, by, a silicon mat: (13). In this case the pressure is ~o actually directed to those parts of the multiwell plate; (1) which are supported by the top surface of the heat block (4) (or by parts of the top surface surrounding individual wells depending on the geometry of the heat block) and not to the thin walls of the wells of the plate as it is the case for the PCR tubes or conventional PCR plates. This advantage makes it possibe to increase the sealing pressure of t;he heated lid (10) several fold when ~5 compared to the conventionally used pressure of 30-50 g per well without cracking the conically shaped walls of the wells (2).
The extremely thin walls of the wells, i.e. 20-~40 microns, are highly flexible~as the multiwell plates are thermoformed out of highly elastic films (or sheets depending on the draw ratio). The walls of the wells are highly resistant against stress cracking, due to zo their flexibility and elasticity. As the wells of the plate, positioned on the heat block, are tightly sealed at room temperature, the air pressure in the wells will increase at elevated temperatures. The increased air pressure causes a deformation of the wails of wells and brings them in tight thermal contact with the surface of the walls of the individual sample wells machined in the body of the heat block. Standard PCR plates {having relatively z5 thick and rigid walls of.the wells) require that the co~ucally shaped walls of the wells have to match perfectly with the shape of the wells machined in the body of the heat block to guarantee a close thermal contact (see for example U.S. Patent No 5,475,610).
This requirement is not as critical for the ultrathin walled multiwell plates of the invention, due to flexibility and elasticity of the walls of the wells. Using this advantage, special shapes 30 of both, the walls of the wells of the plate and the wells of the heat block can be differently designed. These differently designed wells can promote an even closer thermal contact after positioning the plate into the heat block.
Another aspect of the invention concerns the frame of the multiwell plates. As the plates can be formed of very thin films (depending on the. draw ratio of the well; supra) the flexibility of, for example, standard-format plates, i.e. 96-well PCR (8;5 x 12,5 cm) plates, is such that handling is not easily possible anymore. Therefore, depending on the s geometry of the plate, a supporting frame might be needed, for example for industry standard formats, i.e. 96-, I92-, 384-well PCR plates. This frame can support, for example in case of small plates, the edges of the plate, or individual wells of the plate, or groups of wells. For handling with robots, for example, the frame can be injection molded in the form of the standard skirted microplates containing the array of holes in the top vo surface of the frame matching the array of wells of the ultrathin multiwell plate. The plate can be attached to the frame by for example heat bonding. However, for small format plates including the frame can be formed as a single piece by using specially designed moulds.
The polypropylene-based plastics are PCR-compatible and therefore widely used ~s for injection molding of PCR tubes and/or multiwell plates. In addition, they are resistant to stress cracking and have a reduced water vapor sorption when compared to other plastics (e.g. polycarbonate). Such plates can be thermoformed in both, standard industry formats, i.e. 96-, 192- and 384-well PCR plates for large scale applications, supported by robots and small foot-print formats to match small foot-print thermocyclers, i.e. "personal zo thermocyclers".
The following example serves to illustrate the invention but should not be construed as a limitation thereof.
2s Example:
Fig.l illustrates a 36-well ultrathin walled multiwell plate according to the invention. The plate was designed for rapid temperature cycling of saunples ranging from 0.5-4 p.l using a small foot-print pettier-driven heat block thermocycler supplied with a "wine-press" type heated lid (Fig. 2). The volume of the wells is 16 p,l and the distance between the wells is 30 4.5 mm, i.e. industry standard for high sample density 384-well PCR plates.
The diameter of the openings of the wells is 3.8 mm and the height ~of the wells is 3 mm.
The average thickness of the walls of the wells is 30 p,m. The frame (3) was cut out of a polypropylene sheet of a thickness of 0.5 mm and heat bonded to the plate (1). The area of the plate (1) WO flfl/2S92fl PCT/EP99/08t78 is 30 x 30 mm. As shown in Figure 1, the handling of the plate (1) containing the multiple wells (2) is facilitated, by a rigid 0.5-1 mm thick plastic frame (3) which is heat bonded to the plate. As shown in Figure 2, the frame (3) is noi; in direct thermal contact with the block (4) during thermocycling because the inner contour (5) of the frame (3) matches the s outer contour (6) of the heat block (4) of the thermocycler (7 =
thermoelectric heat pump and 8 = air-forced heat sink).
The ultrathin walled multiwell plate according to the invention (Fig. l } was experimentally tested for the amplification of a 455=base pairs long fragment of human ~o papilloma virus DNA. The sample volume was 3 pl. For various PCR reactions, the average romping rate of the thermo cycler was varied from 4° C to 8° C per second. The samples (i.e. standard PCR-mixtures without any carrier molecules) were transferred into the wells of the plate by means of conventional pipelaing equipment. The plate was covered by standard sealing film (Microseal A; MJ-Research, USA); transferred into the ~s heatblock of the thermocycier and tightly sealed by the heated lid as shown in Fig. 2.
Upon sealing, a number of 30 PCR cycles was performed in 15-25 minutes depending on the romping rate of the thermo cycler. The PCR product was analyzed by conventional agarose electrophoresis. The 455-base pairs long DNA fragment was amplified with a high specificity at the indicated romping rates (supra).
2o Plates according to the invention with well volumes of 35 pl were successfully tested for temperature cycling of samples of a volume of 20 ~I. Thereby, 30 PCR cycles were performed in 20-30 minutes at a romping rate of 6° C per second.
Surprisingly, although the average thickness of the walls was 20 rn~icrons and the volume of the wells was 35 pl, samples of a volume of as few as O.S p,l can be easily amplified without zs reducing the PCR efficiency.
In conclusion, the ultrathin walled multiwell plates according to the invention, allow a simple and rapid loading of multiple samples by conventional pipettes, rapid sealing of all samples by using conventional sealing i~lms and rapid DNA
amplification 30 (15-30 minutes for 30 cycles) with an improved spec:ifcity typical for rapid cycling (Wittwer et al., Analytical Biochem:, 186, 328-331 [1990]) using appropriate heat block thermocyclers (i.e. romping rate in the range of 4° C to 8° C
per second).
Temperature cycling of biological samples is a central moment in DNA
amplification by the palymerase chain reaction (PCR:) (Saiki et al., Science, 239, 487-491 [1988]). Much effort is being expended in developing various alternative reactors and technologies for rapid temperature cycling of small-volume samples (Kopp et al., Science ~5 280, 1046-1048 [1998]; Belgrader et al., ].Forensic Science 43, 3i5-319 [1998]; Wittwer et al., Analytical Biochem., 186, 328-331 [1990] and U.S. Patent No 5,4SS;17S;
Woolley et al., Analytical Chem., 68, 4081-4086 [(i996]).
One commercially available type of microreactor and thermocycler for rapid temperature cycling of small samples is a glass capillary tube and a hot-air thermocycler zo from Roche Molecular Biochemicals (cat No. 1909 ?.39 and cat No. 20I 1468, respectively).
The glass capillary tube can hold reaction volumes ranging from 10 to 20 p.l.
The hot-air thermocycler can hold 32 capillaries and perform 30 - 40 PCR cycles in 20-30 minutes.
However, these rapid DNA amplification technology is connected with various disadvantages, for example:
25 a) The handling of the individual capillaries is relatively cumbersome.
b) The relatively large glass surface adsorbs components of the standard PCR-mixtures.
This might inactivate the reaction. Therefore, various carrier molecules, i.e.
proteins or even DNA, must be added and the concentrations of the components reoptimized.
c) The cost of the capillary tube, as a disposable PCR container, is high when compared 3o to the standard 0.2 ml PCR tube.
d) The experimental throughput using this system is limited.
It is surprising that only little research has been conducted to improve the basic performance in sample size and speed of the widely used, conventional heat block thermocycling of samples contained in plastic tubes or multiwell plates. One known improvement of heat block temperature cycling of samples contained in plastic tubes has been described by Half et al. (Biotechniques, 10, lOEi-112, j1991] and U.S.
Patent No 5,475,610). They describe a special PCR reaction-compatible one-piece plastic s microcentrifuge tube, i.e. a thin-walled PCR tube. The tube has a cylindrically shaped upper wall section, a relatively thin (i.e. approximately 0.3 rnm) conically-shaped lower wall section and a dome-shaped bottom. The samples as small as 20 p,1 are placed into the tubes, the tubes are closed by deformable, gas-tight caps and positioned into similarly shaped conical wells machined in the body of the heat block. The heated cover ~o compresses each cap and forces each tube down firmly into its own well. The heated platen (i:e. heated lid) serves several goals by supplying the appropriate pressure to the caps of the tubes: it maintains the conically shaped walls in close thermal contact with the body of the block; it prevents the opening of the caps. by increased air pressure arising in the tubes at elevated temperatures. In addition, it maintains the parts of the tubes that ~s project above the top surface of the block at 95° -100° C in order to prevent water condensation and sample loss in the course of thermocycling. This made it possible to exclude the placing of mineral oil or glycerol into the; wells of the block in order to improve the heat transfer to the tubes and the overlaying of the samples by mineral oil that prevented evaporation but also served as added tlhermal mass. In addition, the PCR
zo tubes can be put in a two-piece holder (US patent 5,710,381) of an 8x12, 96-well microplate format, which can be used to support the high sample throughput needs with any number between 1 and 96 individual reaction tubes.
In DE 4022792 the inventors describe a plate with cylindrically shaped walls of the wells and spherically shaped bottoms thereof. ThE; individual wells of the plate were zs formed by melting a polycarbonate sheet in the range of 0.27-0.5 mm by a stream of hot air. This technology leads to relatively thin walls in the range of 0.08-0.2mm. The biological samples were placed into the wells, covered with polycarbonate film (0. i mm) and the individual wells were thermosealed by a special press. Upon sealing the plate was placed on the thermoblock and fixed by screws. Though theoretically the heat transfer to 3o the samples is improved, however, the way of positioning the plate on the block and the cylindrical and spherical geometry of the well prevent a close thermal contact with the heating block. During thermocyling, due to the large thermal expansion, the plate fixed by WO OOI25920 PCTl~P99/0817g screws becomes deformed and the close thermal contact is not maintained anymore.
Therefore, by using the above technology rapid cycling reactions cannot be performed.
The other known improvement of heat block thermocycling is described in PCT
patent application WO 98/43740. It concerns a neat block thenmocycler with an increased ramping rate, i.e. 4° Clsecond). The thermocycler ca~;~ hold 96 PCR
tubes (each of a volume of 0.2 ml) or 96-well PCR plates. Theoretica,lIy, the thermocycler can perform 30 PCR cycles in 20-30 minutes, provided that only a fe;w seconds are spent to reach the temperature equilibrium between the heat block and the samples.
However, as described in U.S. Patent No 5,508,197, even if the temperature of the ~o heat-transfer media; i.e. water, is changed almost instantaneously, it takes approximately 15 seconds to reach equilibrium between water and t:he 15-20 p.l samples in the standard PCR plates. This means that for 30 PCR cycles approximately 20 minutes are spent to reach the equilibrium between heat-transfer media ar.~d the 15-20 ul samples in the plates.
In comparison, the above mentioned heat block cycler (WD 98/43740) operating ~s at a ramping rate of 4° C/second, needs for the heat-block temperature transitions during 30 PCR cycles 10 minutes only. This shows that the :major limiting factor for rapid temperature cycling of small samples in platic PCR tubes or PCR plates is the low efficiency of the heat transfer through the walls of conventional PCR tubes or plates, respectively.
zo The present invention concerns plastic multiwell plates for performing heat block thermocycling of multiple samples. More specifically, it concerns ultrathin-walled multiwell plates with an improved heat transfer to small samples. Ultrathin-walled multiwell plates are suited for rapid, oil-free, heat block temperature cycling of small-volume samples (i.e. approximately 1-20 p,l), wherea:> the Lower limit is given by the zs reliability of the conventional pipetting systems.
Figure 1 illustrates an example of a multiwell plate according to the invention.
Figure 2 illustrates the positioning of the plate in the )block of the thermal cycler.
One aspect of the present invention concerns the considerably decreased thickness (i.e. approximately 7.5-15 fold) of the well walls when compared to known thin-walled 3o PCR tubes (U.S. Patent No 5,475,610.). This can be reached, for example, by means of thermoforming the plates out of thin thermoplastic films. Such thermoplastic films are, for example, polyolefin films, such as metallocene-catalyzed polyolefin films and/or copolymer films. Usually, the multiwell plate is vacuumformed out of cast, unoriented polypropylene fzlm, polypropylene-polyethylene copolymer films or metallocene-catalyzed polypropylene films. The film is formed into a negative ("female") mould comprising a plurality of spaced-apart, sonically shaped wells which are machined in the body of a mould in the shape of rectangular- or square-array. The thickness of the film s for vacuumforming sonically shaped wells is chosen according to the standard rule used for thermoforming, i.e. thickness of the film = well draw ratio x thickness of the wall of the formed well.
For example, vacuumforming wells with a draw ratio of two and an average thickness of the walls of 30 microns results-in a film thickness of 60 microns. The ~o average optimum wall thickness was found to be 20-~40 microns. The thickness of the well is reduced 7.5-15 fold when compared to the wall thickness of the formerly improved PCR tube desribed in U.S. Patent No 5,475,610. Using the Fourier equation for heat transfer and the equation for temperature transfer through solid substances, it can be shown that heat transfer through one square millimeter of the surface of the well of the ~s plate is increased 7.5-15 fold and the time of temperature transfer through the wall is decreased 56-225 fold when compared to the said PC'R tube. This drastic decrease in time can be explained by the fact that the time needed for ahe transfer of temperature front is proportional to the square power of distance. It can be easily calculated that the time of the temperature transfer through the ultrathin walls of the mufti-well plate is in the range Zo of milliseconds, whereas for the said PCR tube (LT.S. Patent No 5,475,610) it is in the range of seconds. This explains the well known fact that thin (20-40 microns) plastic films are poor thermo insulators.
The thickness of the walls of the formed well:. is gradually reduced to the bottoms of the wells due to vacuumforming of the wells into a~ negative mould. This geometry of zs the walls of the wells provides several advantages:
~ The relatively thick upper parts of the walls of the wells cause additional rigidity of the whole multiwell plate.
~ During heating of the heat block of the thermocycl.er, a vertical temperature gradient is formed in the sample, due to the gradient of the wf;ll-wall thickness. This vertical 3o temperature gradient causes intensive convective mixing of the sample in sonically shaped wells and increases the heat transfer through the sample. In comparison, this convective mixing of the sample is much less eff dent in conventional PCR
plates/tubes with a uniform wall thickness.
Another aspect of the invention concerns the height of the wells of the rnultiwell plate. The height of the conically shaped wells is equal to the height of the similarly shaped sample wells machined in the body of the heat block. Thus, this geometry of the wells (2) enables the positioning of the plate (1) on i:he heat block (4) as shown in Figure s 2. As shown (Figure 2), in contrast to the conventional PCR plates, the walls of the wells (2) of the mufti-well plate (1) do not project above tile top surface of the block {4). The type of positioning provides several advantages: Thc; pressure caused by the screw (12) to the lid (10) {heating element (11)) can be increased i.n order to obtain efficient sealing of the samples (9) sealed, for example, by, a silicon mat: (13). In this case the pressure is ~o actually directed to those parts of the multiwell plate; (1) which are supported by the top surface of the heat block (4) (or by parts of the top surface surrounding individual wells depending on the geometry of the heat block) and not to the thin walls of the wells of the plate as it is the case for the PCR tubes or conventional PCR plates. This advantage makes it possibe to increase the sealing pressure of t;he heated lid (10) several fold when ~5 compared to the conventionally used pressure of 30-50 g per well without cracking the conically shaped walls of the wells (2).
The extremely thin walls of the wells, i.e. 20-~40 microns, are highly flexible~as the multiwell plates are thermoformed out of highly elastic films (or sheets depending on the draw ratio). The walls of the wells are highly resistant against stress cracking, due to zo their flexibility and elasticity. As the wells of the plate, positioned on the heat block, are tightly sealed at room temperature, the air pressure in the wells will increase at elevated temperatures. The increased air pressure causes a deformation of the wails of wells and brings them in tight thermal contact with the surface of the walls of the individual sample wells machined in the body of the heat block. Standard PCR plates {having relatively z5 thick and rigid walls of.the wells) require that the co~ucally shaped walls of the wells have to match perfectly with the shape of the wells machined in the body of the heat block to guarantee a close thermal contact (see for example U.S. Patent No 5,475,610).
This requirement is not as critical for the ultrathin walled multiwell plates of the invention, due to flexibility and elasticity of the walls of the wells. Using this advantage, special shapes 30 of both, the walls of the wells of the plate and the wells of the heat block can be differently designed. These differently designed wells can promote an even closer thermal contact after positioning the plate into the heat block.
Another aspect of the invention concerns the frame of the multiwell plates. As the plates can be formed of very thin films (depending on the. draw ratio of the well; supra) the flexibility of, for example, standard-format plates, i.e. 96-well PCR (8;5 x 12,5 cm) plates, is such that handling is not easily possible anymore. Therefore, depending on the s geometry of the plate, a supporting frame might be needed, for example for industry standard formats, i.e. 96-, I92-, 384-well PCR plates. This frame can support, for example in case of small plates, the edges of the plate, or individual wells of the plate, or groups of wells. For handling with robots, for example, the frame can be injection molded in the form of the standard skirted microplates containing the array of holes in the top vo surface of the frame matching the array of wells of the ultrathin multiwell plate. The plate can be attached to the frame by for example heat bonding. However, for small format plates including the frame can be formed as a single piece by using specially designed moulds.
The polypropylene-based plastics are PCR-compatible and therefore widely used ~s for injection molding of PCR tubes and/or multiwell plates. In addition, they are resistant to stress cracking and have a reduced water vapor sorption when compared to other plastics (e.g. polycarbonate). Such plates can be thermoformed in both, standard industry formats, i.e. 96-, 192- and 384-well PCR plates for large scale applications, supported by robots and small foot-print formats to match small foot-print thermocyclers, i.e. "personal zo thermocyclers".
The following example serves to illustrate the invention but should not be construed as a limitation thereof.
2s Example:
Fig.l illustrates a 36-well ultrathin walled multiwell plate according to the invention. The plate was designed for rapid temperature cycling of saunples ranging from 0.5-4 p.l using a small foot-print pettier-driven heat block thermocycler supplied with a "wine-press" type heated lid (Fig. 2). The volume of the wells is 16 p,l and the distance between the wells is 30 4.5 mm, i.e. industry standard for high sample density 384-well PCR plates.
The diameter of the openings of the wells is 3.8 mm and the height ~of the wells is 3 mm.
The average thickness of the walls of the wells is 30 p,m. The frame (3) was cut out of a polypropylene sheet of a thickness of 0.5 mm and heat bonded to the plate (1). The area of the plate (1) WO flfl/2S92fl PCT/EP99/08t78 is 30 x 30 mm. As shown in Figure 1, the handling of the plate (1) containing the multiple wells (2) is facilitated, by a rigid 0.5-1 mm thick plastic frame (3) which is heat bonded to the plate. As shown in Figure 2, the frame (3) is noi; in direct thermal contact with the block (4) during thermocycling because the inner contour (5) of the frame (3) matches the s outer contour (6) of the heat block (4) of the thermocycler (7 =
thermoelectric heat pump and 8 = air-forced heat sink).
The ultrathin walled multiwell plate according to the invention (Fig. l } was experimentally tested for the amplification of a 455=base pairs long fragment of human ~o papilloma virus DNA. The sample volume was 3 pl. For various PCR reactions, the average romping rate of the thermo cycler was varied from 4° C to 8° C per second. The samples (i.e. standard PCR-mixtures without any carrier molecules) were transferred into the wells of the plate by means of conventional pipelaing equipment. The plate was covered by standard sealing film (Microseal A; MJ-Research, USA); transferred into the ~s heatblock of the thermocycier and tightly sealed by the heated lid as shown in Fig. 2.
Upon sealing, a number of 30 PCR cycles was performed in 15-25 minutes depending on the romping rate of the thermo cycler. The PCR product was analyzed by conventional agarose electrophoresis. The 455-base pairs long DNA fragment was amplified with a high specificity at the indicated romping rates (supra).
2o Plates according to the invention with well volumes of 35 pl were successfully tested for temperature cycling of samples of a volume of 20 ~I. Thereby, 30 PCR cycles were performed in 20-30 minutes at a romping rate of 6° C per second.
Surprisingly, although the average thickness of the walls was 20 rn~icrons and the volume of the wells was 35 pl, samples of a volume of as few as O.S p,l can be easily amplified without zs reducing the PCR efficiency.
In conclusion, the ultrathin walled multiwell plates according to the invention, allow a simple and rapid loading of multiple samples by conventional pipettes, rapid sealing of all samples by using conventional sealing i~lms and rapid DNA
amplification 30 (15-30 minutes for 30 cycles) with an improved spec:ifcity typical for rapid cycling (Wittwer et al., Analytical Biochem:, 186, 328-331 [1990]) using appropriate heat block thermocyclers (i.e. romping rate in the range of 4° C to 8° C
per second).
Claims (8)
1. Ultrathin-walled multiwell plate for heat block thermocycling of samples comprising an array of small-volume wells of identical height with the similarly shaped sample wells formed in the top surface of the heat block of the thermocycler, wherein the walls of the wells have an average thickness of 20-40 microns.
2. Ultrathin-walled multiwell plate according to claim 1, wherein the height of the wells of the plate is not more than the height of the sample wells formed in the top surface of the heat block of the thermocycler
3. Ultrathin-walled multiwell plate according to claim 1, wherein the walls of the wells are conically shaped.
4. Ultrathin-walled multiwell plate according to claim 1, wherein the thickness of the walls of the wells decreases from top to bottom.
5. Ultrathin-walled multiwell plate according to claim 1, wherein the wells of said multiwell plate are thermoformed into negative mould.
6. Ultrathin-walled multiwell plate according to claim 1, wherein the walls of the wells are deformable.
7. Ultrathin-wailed multiwell plate according to claim 1, wherein the said microwell plate comprises a rigid supporting frame.
8. Ultrathin-walled multiwell plate according to claim 1, wherein the volume of the well is in the range of 16-85 µl.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP98120187.4 | 1998-10-29 | ||
| EP98120187A EP1000661A1 (en) | 1998-10-29 | 1998-10-29 | Ultrathin-walled multiwell plate for heat block thermocycling |
| PCT/EP1999/008178 WO2000025920A1 (en) | 1998-10-29 | 1999-10-28 | Ultrathin-walled multiwell plate for heat block thermocycling |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2348564A1 true CA2348564A1 (en) | 2000-05-11 |
Family
ID=8232855
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002348564A Abandoned CA2348564A1 (en) | 1998-10-29 | 1999-10-28 | Ultrathin-walled multiwell plate for heat block thermocycling |
Country Status (6)
| Country | Link |
|---|---|
| EP (2) | EP1000661A1 (en) |
| JP (1) | JP4538152B2 (en) |
| AT (1) | ATE257743T1 (en) |
| CA (1) | CA2348564A1 (en) |
| DE (1) | DE69914220T2 (en) |
| WO (1) | WO2000025920A1 (en) |
Families Citing this family (19)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6780617B2 (en) | 2000-12-29 | 2004-08-24 | Chen & Chen, Llc | Sample processing device and method |
| US7799521B2 (en) | 1998-06-24 | 2010-09-21 | Chen & Chen, Llc | Thermal cycling |
| AU4957699A (en) | 1998-06-24 | 2000-01-10 | Chen & Chen, Llc | Fluid sample testing system |
| EP1045038A1 (en) * | 1999-04-08 | 2000-10-18 | Hans-Knöll-Institut Für Naturstoff-Forschung E.V. | Rapid heat block thermocycler |
| US7347977B2 (en) | 2000-06-08 | 2008-03-25 | Eppendorf Ag | Microtitration plate |
| US6660232B1 (en) * | 2000-09-29 | 2003-12-09 | Promega Corporation | Multi-well assay plate and plate holder and method of assembling the same |
| EP1427531B1 (en) | 2001-09-11 | 2016-10-19 | Iquum, Inc. | Sample vessels |
| EP1603674B1 (en) | 2003-02-05 | 2016-01-06 | Iquum, Inc. | Sample processing |
| US7442542B2 (en) | 2003-03-24 | 2008-10-28 | Agency For Science, Technology And Research | Shallow multi-well plastic chip for thermal multiplexing |
| GB0701999D0 (en) * | 2007-02-02 | 2007-03-14 | Advanced Biotech Ltd | Multi-well improved plate |
| DE102007062441A1 (en) | 2007-12-20 | 2009-06-25 | Aj Innuscreen Gmbh | Mobile rapid test system for nucleic acid analysis |
| US9034635B2 (en) * | 2008-02-20 | 2015-05-19 | Streck, Inc. | Thermocycler and sample vessel for rapid amplification of DNA |
| US8802000B2 (en) | 2008-08-01 | 2014-08-12 | Bio-Rad Laboratories, Inc. | Microplates with ultra-thin walls by two-stage forming |
| FR2941876B1 (en) * | 2009-02-06 | 2012-12-07 | Bio Rad Pasteur | THERMAL VALIDATION APPARATUS, ASSEMBLY OF A DEVICE FOR PROCESSING BIOLOGICAL SAMPLES AND SUCH APPARATUS, AND METHOD FOR MANUFACTURING SUCH APPARATUS |
| EP2404672A1 (en) * | 2010-07-06 | 2012-01-11 | Universiteit Twente | High troughput multiwell system for culturing 3D tissue constructs in-vitro or in-vivo, method for producing said multiwell system and methods for preparing 3D tissue constructs from cells using said multiwell system |
| WO2012166913A1 (en) | 2011-06-01 | 2012-12-06 | Streck, Inc. | Rapid thermocycler system for rapid amplification of nucleic acids and related methods |
| CA2916990C (en) | 2013-06-28 | 2023-05-23 | Streck, Inc. | Devices for real-time polymerase chain reaction |
| KR102206856B1 (en) * | 2017-12-11 | 2021-01-25 | (주)바이오니아 | Polymerase Chain Reaction System |
| DE102019106699B4 (en) | 2019-03-15 | 2024-01-25 | Analytik Jena Gmbh+Co. Kg | Device and method for the thermal treatment of samples |
Family Cites Families (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2642156B1 (en) * | 1989-01-20 | 1994-05-20 | Bertin Et Cie | METHOD AND DEVICE FOR QUICK REGULATION OF A WALL TEMPERATURE |
| DE4022792A1 (en) * | 1990-07-18 | 1992-02-06 | Max Planck Gesellschaft | PLATE WITH AT LEAST ONE RECESS FOR RECEIVING CHEMICAL AND / OR BIOCHEMICAL AND / OR MICROBIOLOGICAL SUBSTANCES AND METHOD FOR PRODUCING THE PLATE |
| DE4022794A1 (en) * | 1990-07-18 | 1992-02-06 | Max Planck Gesellschaft | METHOD FOR PRODUCING A PLATE WITH AT LEAST ONE TUBE OPEN TO THE TOP FOR RECEIVING CHEMICAL AND / OR BIOCHEMICAL AND / OR MICROBIOLOGICAL SUBSTANCES AND PLATE PRODUCED BY THE METHOD |
| DE4029004C1 (en) * | 1990-09-13 | 1992-04-02 | Max-Planck-Gesellschaft Zur Foerderung Der Wissenschaften Ev, 3400 Goettingen, De | |
| KR100236506B1 (en) * | 1990-11-29 | 2000-01-15 | 퍼킨-엘머시터스인스트루먼츠 | Apparatus for polymerase chain reaction |
| US5601141A (en) * | 1992-10-13 | 1997-02-11 | Intelligent Automation Systems, Inc. | High throughput thermal cycler |
| GB9225307D0 (en) * | 1992-12-03 | 1993-01-27 | Norton David R | Sealing films for multiwell plates,strips & microtubes |
| DK72493D0 (en) * | 1993-06-18 | 1993-06-18 | Risoe Forskningscenter | SOLID SUPPORTS FOR USE IN PEPTIDE SYNTHESIS AND ASSAYS |
| JPH0751099A (en) * | 1993-08-11 | 1995-02-28 | Toyobo Co Ltd | Method for examining sequence of nucleic acid and examination apparatus |
| US5472672A (en) * | 1993-10-22 | 1995-12-05 | The Board Of Trustees Of The Leland Stanford Junior University | Apparatus and method for polymer synthesis using arrays |
| DE4440294A1 (en) * | 1994-11-11 | 1996-05-15 | Boehringer Mannheim Gmbh | System for the incubation of sample liquids |
| DE19534632A1 (en) * | 1995-09-19 | 1997-03-20 | Boehringer Mannheim Gmbh | System for temperature change treatment of sample liquids |
| JP3705606B2 (en) * | 1996-01-25 | 2005-10-12 | ビージェイエス カンパニー リミテッド | heating |
| JPH10117765A (en) * | 1996-10-18 | 1998-05-12 | Ngk Insulators Ltd | Specimen holder and its production |
| DE19739119A1 (en) * | 1997-09-06 | 1999-03-11 | Univ Schiller Jena | Microtitration plate for wide application |
| JP2001252067A (en) * | 1998-09-22 | 2001-09-18 | Sumitomo Bakelite Co Ltd | Multiwell plate for freezing cultured cell |
-
1998
- 1998-10-29 EP EP98120187A patent/EP1000661A1/en not_active Withdrawn
-
1999
- 1999-10-28 EP EP99952630A patent/EP1133359B1/en not_active Expired - Lifetime
- 1999-10-28 WO PCT/EP1999/008178 patent/WO2000025920A1/en active Search and Examination
- 1999-10-28 JP JP2000579350A patent/JP4538152B2/en not_active Expired - Fee Related
- 1999-10-28 CA CA002348564A patent/CA2348564A1/en not_active Abandoned
- 1999-10-28 AT AT99952630T patent/ATE257743T1/en active
- 1999-10-28 DE DE69914220T patent/DE69914220T2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| EP1000661A1 (en) | 2000-05-17 |
| EP1133359B1 (en) | 2004-01-14 |
| JP4538152B2 (en) | 2010-09-08 |
| EP1133359A1 (en) | 2001-09-19 |
| JP2002528108A (en) | 2002-09-03 |
| WO2000025920A1 (en) | 2000-05-11 |
| ATE257743T1 (en) | 2004-01-15 |
| DE69914220D1 (en) | 2004-02-19 |
| DE69914220T2 (en) | 2004-11-11 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Discontinued |