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Title:
THIN WALLED MICROPLATE
Document Type and Number:
WIPO Patent Application WO/2019/092390
Kind Code:
A1
Abstract:
A thin-walled microplate suitable for use in the Polymerase Chain Reaction (PCR) technique comprising a plurality of thin-walled tubes or wells arranged in a fixed array, each well having an upper portion with an open top and a lower, frustoconical portion having a substantially flat bottom.

Inventors:
DAY, Paul Francis (The Aldermoor, Holmbury St MaryDorking, Surrey RH5 6NR, GB)
Application Number:
GB2018/000144
Publication Date:
May 16, 2019
Filing Date:
November 12, 2018
Export Citation:
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Assignee:
4TITUDE LTD (The North Barn, Surrey Hills Business ParkDamphurst Lan, Wotton Surrey RH5 6QT, GB)
DAY, Paul Francis (The Aldermoor, Holmbury St MaryDorking, Surrey RH5 6NR, GB)
International Classes:
B01L3/00; C12M1/00; G01N35/00
Domestic Patent References:
WO2017169196A12017-10-05
WO2014143044A12014-09-18
Foreign References:
US20100248213A12010-09-30
US5670118A1997-09-23
CN103240135A2013-08-14
Attorney, Agent or Firm:
WHITAKER, Iain (Alban Patent Agency, The Old Pump House 1a Stonecross,St Albans, Hertfordshire AL1 4AA, GB)
Download PDF:
Claims:
CLAIMS

1. A thin-walled microplate suitable for use in the Polymerase Chain Reaction (PCR) technique comprising a plurality of thin-walled tubes or wells arranged in a fixed or rancom access array, each well having an upper portion with an open top and a lower, frustoconical portion having a substantially flat bottom

2. A thin-walled m croplate according to Claim 1 wherein the surface of the substantially flat bottom of each of the wells in the array are in substantially the same horizontal plane such that the inner bottom surface of each well i the array is substantially equidistant from the top of the microplate, in order that items sucn as cells in the bottom of different wells in the array are ir substantially the same focal plane when viewed from above through -he opening n the top cf the respective wells.

3. A thin-walled microplate according to Claim 2 wherein the distance to the bottom of each well is the same to within +/- 0.1 mm.

4. A thin-walled nicroplate according to Claim 2 or Claim 3 wherein the distance to the bottom of each well is the same to within +/- 0.05mrr or better.

5. A thin-walled microplate according to any preceding Claim wherein the external diameter of the flat bottom surface of each well is in the range 2.0mm \o 4.5nm.

6. A thin-walled microplate according to Claim 5 wherein the external diameter of the flat bottom s jrface of each well is in the range 2.5mm to 3.5mm. 7. A thin-walled microplate according to any preceding Claim wherein the substantially flat bottom surface of one or more wells in the array incorporates a machine readable code.

8. A thin-walled microplate according to Claim 7 wherein the machine readab'e code is readab'e using an optical vision system.

9. A thin-walled microplate according to Claim 7 or Claim 8 wherein the machine readable code is on the external surface of the substantially flat bottom. 10. A thin-walled microplate according to any preceding Claim wherein the external surface of the flat bottom on each well incorporates a protection: ring or bead.

11. A thin-walled microplate according to Claim 10 wherein the protection ring/bead is located substantially on the outer circumference of the flat bottom.

12. A thin-walled microplate according to any preceding Claim wherein the substantially flat bottcm of each well is formed from substantially clear material such that each well has a flat, optically-clear window.

13. A thin-walled microplate according to any preceding Claim wherein the array of thin-walled tubes or wells are held in a substantially rigid frame. 14 A thin-walled microplate according to Claim 13 wherein the substantia ly rigid frame is formed from a first plastics material and the tubes or wells are formed From a second plastics material that is suitable for PCR use.

15. A thin-walled microplate according to Claim 13 or Claim 14 wherein the wells are not integrally (permanenily) fixed in the rigid frame but can be individually removed from the frame and are therefore randomly accessible.

16. A thin-walled microplate acco-ding to Claim 13 or Claim 14 wherein the wells are substantially permanently fixed in the rigid frame and are therefore not randomly accessible.

Description:
THIN WALLED MICROPLATE

Field of the Invention

The present inventior -elates to thin-walled nicroplates for use in Polymerase Chain Reaction |PCR) reactions, compr sing an array of thin-walled tubes or wells adapted for use in a thermal cycler. It is particular y applicable to an array of thin-walled tubes having open tcps and lower, frustocoiical portions having a substantially flat bottom. Background to the Invention

A typical human cell consists of about 6 billion base pairs of DNA and 600 million bases of mRNA. Usually a mix containirg millions of cells is used in sequencing the DNA or RNA using traditional methods However by using Next Generation Sequencing (NGS) of DNA and RNA from a single cell, cellular functions can be investiga ed. In order to carry cut expe r iments on a single cell the following steps are required: isolation of a single cell, rxcleic acid extraction and amplification, sequencing library preparation, sequercing and bioinformatic data analysis. Needless to say, it is much more challeng ng to perorm single cell sequencing in comparison with sequencing from cells h bulk. The minimal amount of starting materials from a single cell means that degradation, sample loss and/or contamination can cause pronounced efects on the quality of sequencing data. Nonethe ess, recent technical improvements make single cell sequencing a promising tool for approaching otherwise intractable problems. There is currently no standardized technique for single-cell isolation. Individual cells can be collected by micromanipulation, for example by serial dilution or by using a patch pipette or nanoiube to harvest a single cell. Currently this separation step is generally carried out in sample tubes in which the cell(s) can be viewed, but which are inherently unsu table for use in a PG thermal cycler. Only once the operator has determined that only the desired single cell is present is this then transferred to a PCR t jbe or well for PCR anc sequencing. This is because known PCR wells are not suitable for harvesting ard viewing a single cell. This transfer from the harvesting tube to a PCR well requires an additional manipulation and can easily give rise to the problems of degradatioi, sample loss and/or contamination referred to above. A further problem wit current FCR wells is how to apply a marking to a PCR well to give it a unique identity. A cap to the PCR tube can be labelled, but caps can become detached, and this is taot the same as labelling the tube itself. The upper part of the outside of a PCR well can be labelled but this involves producing a label on a tightly curved surface, making the machine reading of any label problematic. Such machine reading becomes impossible if the PCR well in question is in the middle of an array of wells, for example a 12 by 8 aray of 95 tubes or wells.

It is an object of the present invention to overcome or mitigate some or all of the disadvantages outlined above.

Summary of the Invention

According to a first aspect of :he present invention there is provided a thin-walled microplate according to Claim 1. For example there is described a t'nin-walled microplate suitable for use the Polymerase Chain Reaction (PCR) technique comprising a plurality of thin-walled tubes or wells arranged in a fixed or random access array, each well having an upper portion with an open top ard a lower, frustoconical portion having a substantially flat bottom. By providing a flat bottomed well in an array of wells suitable for use in the PCR technique a wide variety of new possibilities are created. It w I be understood tha the bottom to the well will have an internal surface, inside the well, and an external surface on the outside of the well. The bottom surface of the well can be made substantially planar, and horizontal when stood on a horizontal surface, such that a single eel' in the tube can be viewed through ihe top of the tube. If the flat bottom surface of the well is made substantially transparent, i.e incorporates an optically clear window, then a single cell, or some other Feature such as fluorescence within the well, can be viewed from below the well through this optically window, as well as from above. Or alternatively the bottom surface, preferably the external bottom surface, may carry some form of label, preferably in the form of a unique machine readable code.

Preferably the bottom surface of each of the welts in the array are in substantially the same horizontal plane such that items such as a cell or cells in the bottom of different wells in the array are in substantially ths same focal plane when viewed from above through the opening in the top of the respective weHs, or fnm below in the case of a well wi:h a substantially transparent flat bottom window. By 'in substantially the same plane is meant that the distance to the bottom of each well is the same to within +/- 0.1 mrr and more preferably with n +/- 0.05mrr or be:ter.

Preferably the external diameter of the flat bottom surface or portior o" each well is in the range 2.0mm to 4.5mm, more preferably in the range 2.5m m to 3.5mm, with a particularly preferred diameter being 3mm.

Preferably the substantially - " lat bottom surface of one or nore wells in a random access array includes a machine readable code, and more prefera ly suostantially all the wells in the randomly accessible array carry such a cede.

Preferably the machine readable code is readable using an optical vision system sometimes referred to as a Machine Vision (MV) System. Preferably the machine readable code is on the exterral surface of the substantially flat bottom surface of the we I.

In a particularly preferred embodiment the flat bottom surface or window of each well incorporates a protection ring or bead, and preferably the protection ring/bead is located substantially on the outer bottom peripheral crcumference of the flat bottom surface or window. This bead, ring or downstand serves to protect the flat bottom surface of the well from accidental damage caused by, for example, solvents cr abrasion. Contact with solvents for example might degrade any code or other label on the bottom of the well. Any abrasion would detract from optical measurements made through an otherwise substantially optically transpa r ent window, or interfere with the 2D code reading Keeping the flat bottom exterior surface of the well distanced away from poten al contaminants is an important feat jre of the invention.

Preferably the substantia ly flat bottom surface cf each well is fonmed from substantially clear material such that each well has εη optically-clear window. This feature, if present, provides the advantages set out above.

Preferably the array of thin-walled tubes or wells a-e he d in a substantially rigid frame, and this frame may oe formed from a different plastics materials to the wells, such that the substantially rigid frame is formed from a first plastics material and the tubes or wells are formed from a second plastics material that is suitable for PCR use. The wells may be permanently fixed in the frame or may be individually removable from the frame and therefore randomly accessible.

Brief Description of the Drawings

Preferac embodiments of the present invention will now be described by way of example only with reference to the accompanying Figures wherein:- Figures 1A to 1 D show side e evation, cross-secttonal, bottom elevation and perspective views respectively of a single thin-walled PCR well;

Figure 2 shows a perspective view of a single thin-walled PCR well as shown in Figure 1 with a 2D machine readable code on the bottom of the well;

Figure 3A to 3 H show top elevation, side cross-section along C-C, side cross- section along B-B, an end elevation, a side elevation, a partial view frcm underneath, detail F from Figure 3B, and a perspective view from above to one side of a 93 well random access array of the type of wells shown in Figure 1 in a frame.

Description of the Preferred Embodiments

Figu'e 3 illustrates various views of one embodiment of a thin-walled microplate according to the present invention, in this example a microplate containing a 12X8 array of 96 wells. A 12X8 array is given here by way of example only, and other arrays having different configurations and numbers cf wells are possible. Figures 1A tc 10 show various views of one of the individual thin-walled wells used in the array of wells shown in Figure 3. The thin-walled taction wells shown in more detei' in Figure 1 are designed specifically for use in performing Polymerase Chain Reaction (PCR) experiments in a thermal cycler. Eacn well comprises a hollow tube 10 having an opening 11 a: a first end, a short substantially straight sided upoer seel ion 18 adjacent to the open end, and a frustoconical section 12 which is closed in a fluid-tight fashion by a substantially flat bottom portion 13, having internal and external bottom surfaces. The wells are formed from a plastics material with good heat transfer properties and whici is suitable for PCR, such as polypropylene.

It is important that the bottom of each well is substantially flat and substantially horizontal when the microplate is positioned on a horizontal surface. It is also important that the inner bottom surface of each well in an array is substant ally equidistant from the top of the microplate. By substantially equidistance it is meant that the distance to the bottom of each well is the same within +/- 0.1mm, and preferably +/- 0.05mm or bettsr. This tolerance arrangement means that an operator can view an item, such as a single cell, resting on the flat bottorr surface of any of the wdls through an optical imaging system, without having to alter the fecal plane of the instrument. This significantly speeds up the process of checking the content of each wall.

The flat bottom surface may advantageously be made substantially optically clear or transparent, :hus creating a substantially clear optical window in the bottom of the well. One method of achieving this high transparency is to highly polish tie opposing surfaces of the moulding tool that form the bottom of each well. In t is way visual observations may also be made from underneath the m cropiate as 'well as from above. Observation of other effects such as colour changes or fluorescence from the contents of a well may also be made from above or belo the microplate. This is the fist tine this has been possible in an array of PCR wells n a microplate. In a further smbediment, -ather than having an optically clear window or the bottom of a well, ths flat-bottom surface or portion can carry some form of abel; or maen ne readable cede. One example of this is shown in Figure 2 which snows a 2D cede 117 on the bottom of a well. There are a wide variety of known ways to apply machine readable data o the bottom of a test tube, but not a PCR well. For example, US 6,372,2938 (Matrix Technologies Corp) describes a method of applying a multi-layer coating onto a planar exterior surface of a test tube and removing portions of the outer layer. US 6,270,728B (Micronic B.V) describes a process of aser burning a code onto the base of a test ~ube. A further method is to use a treatment tor example a corona treatment, to change the properties o" the exterior of * Jie flat-bottom surface of each well. A machine readable code can then be applied directly onto the bottom of each treated wed. This is a particularly cost effective methoc of applying a code to the bottom of each well.

The provision ol PCR tuces with unique machine readable codes represents a -eal advance fo" experimenters, particularly when those tubes are in a random access array as illustrated in Figure 3. In this example the individual tubes 210 are not integrally (permanently) fixed in the rigid frame 201 , 202 but can be individually removed from the frame and are therefore randomly accessible. That is to say, an operator c=n pish any one tube upwards out of the frame in order to process that particular tube without disturbing the other 95 tubes that are still held capti e in the frame. t will be appreciated that in an alternative embodiment the lubes nay be integrally permanently) fixed into a rigid frame, if that is the fonrnat preferred by the experimenter. Methods of making integrally (permanently) f xed arrays of wells are known, such as those described in US 6,340,589B1 (Eppendorf). '

Whether the outer surface of the bottom of each well is optically clear or carries a (machine readable code, it is important that the surace is orotected in some way. One method of giving a degree of protection is to ncorporate a protection ring cr bead 14, 114 around part of or around the entire cater circumference of the bottom surface, as shown in Figures 18, 1 D and 2. This p-otection ring can be formed as 3 downstand around some of or substantially the entire outer perimeter of the bottom of the well in the moulding process. The protection ring need not be present around the entre circumference of the Dottom cf the well, and may include one or more gaps 15 as shown in Figures 1 D and 2 Furthermore, it need not take the form of a conventional 'ring', but instead cojld take the form of a plurality of individual protrusions se: around the perimeter of the bottom portion. The protection ring, or its equivalent, serves to keep the outer surface of the flat-bottom away from any surface that it might otherwise rest on. Tnis keeps contaminants, such as solvents, away from the machine readable code and also prevents the surface from getting scratched or abraded. Such abrasion would impair the quality of imaging if the bottom or " the well is optically clear, as well as potentially destroy the integrity and readability cf any code.

The diameter of the bottom surface of the frustocorical portion of the -wells is an important feature of this invention. The diameter, which is measured to the inside edge of any protection ring, is preferable 2.0 to 4.5 nm and more preferable 2.5 to 3.5 mm. A particularly preferred diameter for the w ndow in the bottom surface is 3mm ± 10%.

By way of example only of some other typical dimensions the wall thickness of the side wsll of each well in the foistoconical portion is 0.25mm ± 0.325mm, which is a typical wall thickness for carrying out PCR reactions. The thickness of the bottom portion or surface is not critical, as it is not in direct contact with the thermal cycer, and a typical thickness for the bottom of the weH is 0.5m-n. The angle of the side wall of each well in the frustoconical section must correspond to the well angle fo jnd in commercially availab'e thermal cyclers. Typically this angle is 17 degrees. The full well volume of a typical well is in the region of 200 microlitres. The top of each well could incorporate a raised rim or chimney if desired (not shown), to facilitate sealing the wells with some form of sealing strip or film, but this is not essential.

As referred to above, Figure 3 illustrates a thin-walled microplate 200 of the invention, containing an 12 by 8 array of 96 wells of the type illustrated in Figure 1 or Figure 2. As explained above the individual wells 210 are either fixed or removably held captive in a substantially rigid frame made up of a skirt portion 201 and a deck portion 202. In the exsmple iiustrated in Figure 3, the wells are held ir a randomly accessible fashion in the deck portion by a collar 216 and a substantially planar region 218 close to :he top of each well. The substantially planar portion of each well may incorporate a slight indentation in order that the wells are held more tightly in the substantially "igid frame, and are therefore less prone to being dislodged accidenlly during handling. The rigid frame can be made of a first plastics material such as a polycarbonate, a nylon or Acrylonitrile Butadiene Styrene (ABS), and the wells formed from a second plastics material that is suitable for FOR sucn as polypropylene. It wil be understood that new materials do become available over time and the optimum materials for both components will be selected by a materials expert as required.

In summary, in a first version of the present invention there is provided an ar-ay of tubes with highly poHshed flai optical bases, or optical windows, where the wells are substantially permanently fixed in a rigid frame in, for example, a 12X8 array of 96 wells. This arrangement is of interest to scientists who warn to make optical measurements of a tube's contents from either above or below the plate, i.e. by using a laboratory plate reacer as a pre-screen in applications like single cell PCR procedures.

There is a growing trend in the market for scientists to want to do single cell PCR whereby they populate a screening plate that is not a PCR plate but n which each individual tube in the p ate has an optical base and then after correct identification of the desired target cell transfer that cell to a PCR well for lysis and am lification. A plate with an array of PCR wells with flat optical windows in the bottom of each well enables scientists for the first time to screen a plate for single cell morphology and to confirm there is only one cell per well and then use the same plate for performing the PCR. The offers a huge advantage in that the scientist does not have to capture and transfer a single cell from one plate or well to anothe'. with the inherent possibility of degradation, sample loss and/or contamination.

In a second version, wells incorporating optical windows in the 12X8 array of wells are not integrally (permanently) fixed in the rigid frame but can be individually removed from the frame and are therefore randomly accessible. That is to say, an operator can pjsh any one tube out of the frame to process that particular tube without disturbing the other 95 tubes that are still held captive in the frame.

In a third version of the present invention, a flat bottom PCR tube we can be used to carry a 2D code on the flat bottom surface. This 2D coded PCR tube can then be tracked through the laboratory work flow, which is becoming an important feature for DNA storage and for the Molecular Diagnostic market. Thus, flat bottomed PCR tubes with a modified treated bottom surface that has been labelled with a 2D code are held in a randomly accessible array in a substantially rigid frame, such as a 12X8 array of labelled tubes, where the tubes are not integrally (permanently) fixed in the rigid frame but can be individually removed from the frame and are therefore randomly accessible. The key point to labelling all 96 tubes on the underside of the flat bottom surface is that these tubes can be randomly accessed and removed from the array with their code intact. These random accessible 96 tubes may be processed in a 96 well format but can then be accessed individually in downstream processing procedures/experiments. The flat bottom PCR tube with machine readable code on the flat bottom surface can have the code positioned on the flat bottom surface in such a way as to still allow the flat bottom to serve as a flat optical window for readings.