US20080257882A1

US20080257882A1 - Vessel for accurate analysis

Info

Publication number: US20080257882A1
Application number: US12/105,215
Authority: US
Inventors: Bruce R. Turner
Original assignee: Bioinnovations Oy
Current assignee: Thermo Fisher Scientific Oy
Priority date: 2007-04-20
Filing date: 2008-04-17
Publication date: 2008-10-23

Abstract

Vessels used to contain reaction mixtures and that allow for accurate optical or visual detection are described. Vessels that are an integrated tube- and cap assembly connected via one or more hinge straps or devices to other vessels or caps and/or tubes and caps with different light transmitting properties are also described.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates generally to vessels used to contain reaction mixtures and allowing for accurate optical or visual detection. More specifically the invention relates to a family of those vessels known as tube strips with attached caps and single tubes with attached caps.
2. Description of Related Art
Small amounts of reaction mixtures are commonly stored in disposable plastic tubes, which are closed with caps. There are a number of commercially available vessels of this type and accompanying sealing systems, such as microcentrifuge tubes and tubes used, for example, during heating, chilling or thermal cycling (e.g. from ABgene, Applera, Axygen and BIOplastics). The vessels are available in a number of formats including single tubes, tubes arranged in tray type arrays, typically known as multi-well plates, and also strips of attached tubes, typically arranged with a tube center-to-center distance matching that found in one dimension of multi-well plates. The single tubes and strips of tubes are generally sealed by means of a molded cap or strip of caps that fit securely into the mouth(s) of the tube(s). These caps may be separate or integral to the tube(s). It is reasonable to say that the single tubes and strips of tubes are produced exclusively via typical high-pressure injection molding processes.
Because of the good qualities obtained during the manufacture of vessels using high-pressure injection molding, these vessels are suited for applications using optical detection, such as thermal cycling.
The products of a polymerase chain reaction (PCR) are typically analyzed using end point analysis techniques such as gel electrophoresis. These techniques lack speed and accuracy and are useful primarily to determine relative quantities of known and unknown samples and are best used as a simple measure of whether target sequences are present or not.
A next generation family of instruments now exists which enable the user to monitor a PCR process in real time by utilizing protocols involving light excitation and detection. These instruments are generally known as qPCR (quantitative PCR) instruments. While there are many protocols, which may be performed in these instruments, two common ones are known as the SYBR green method and the fluorescent reporter probe method. In the SYBR green method, a DNA binding dye binds to all newly synthesized double-stranded DNA and an increase in fluorescence intensity is measured allowing initial concentrations to be determined. In the fluorescent reporter probe method only a probe sequence is quantified and not all double stranded DNA. It is commonly carried out with a fluorescent reporter and a quencher held in adjacent positions. Upon the breaking of the probe fluorescence may be detected, since more and more of the fluorescent reporter is liberated from its quencher, resulting in an easily detectable increase in fluorescence.
It may be noted that both of these protocols, as well as all conventional qPCR protocols, typically rely upon the introduction of excitation light into the sample inside the vessel by way of passing it through the vessel-sealing device. In the case of strip tubes and single tubes the sealing device is typically a cap. It may further be noted that detection of the emitted signal relies upon the fluorescence being detected through that same sealing device. It may also be noted that, in the instance where many tubes are in close proximity to one another in the thermal cycling device and a light detection protocol is employed, tube opacity is desirable to contain the light signal within the individual tubes thereby increasing detection accuracy. A white tube is particularly advantageous.
The type of vessels generally used in applications such as thermal cycling is available in three different formats:
1. Strips of tubes with attached optical caps, typically strips of either 8 or 12 tubes:

- In the prior art these are always injection molded of one polymer, optionally containing pigment or dye, making the complete assembly either clear or opaque, generally clear. While the product has the benefit of being easy to handle because of the attached cap, this product is less than optimal because one may not procure it for example in an opaque tube/clear cap configuration or a reflective tube/clear cap configuration or a configuration, wherein the tube and the cap both are colored, but with different colors. In some known vessels, the surface of the tube is “frosted”, but this does not provide a completely opaque surface.

2. Single tubes with attached caps:

- In the prior art this form of product is not optimal for the reasons cited above.

3. Strips of tubes without caps:

- Strip tubes without caps are available also in white color. In this format the sealing device is a separate strip of caps made of a clear polymer. While this combination of products will work for applications using optical detection, it lacks the economy and ease of use of the formats with integrated optical caps.

As a general example of available vessel formats, US 20050084957 presents a tube and a method for achieving accurate temperature control for a large number of samples arranged in a microtiter plate format during very rapid thermal cycling PCR protocols.

SUMMARY OF THE INVENTION

It is an aim of the present invention to provide a novel integrated tube-and-cap assembly, wherein the tube and the cap have different optical properties.
Particularly, it is an aim of the present invention to provide a novel vessel for accurate optical detection.
These and other objects, together with the advantages thereof over known vessels and methods, are achieved by the present invention, as hereinafter described and claimed.
The present invention concerns a vessel comprising an integrated tube-and-cap assembly, wherein one or more tube is connected by way of a hinge strap or device to one or more cap.
More specifically, the vessel of the present invention is characterized by what is stated in the characterizing part of claim 1.
Further, the use of the present invention is characterized by what is stated in claims 16 and 17 and the method of the present invention is characterized by what is stated in the characterizing part of claim 18.
Considerable advantages are obtained by means of the invention. Thus, the present invention provides a unique single combined tube and cap structure composed of two separate materials, said combined structure having the economy, utility and ease of use of prior art single material products combined and which may also embody the features of prevention of tube-to-tube light transmission and internal light reflection, thereby improving signal quality
Next, the invention will be described more closely with reference to the attached drawing and a detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a perspective view of the tube-and-cap assembly of the present invention. In FIG. 1 a the assembly of one of the embodiments of the invention is shown. FIG. 1 b is a close-up of the tube portion of the assembly and FIG. 1 c is a close-up of the cap portion.

DETAILED DESCRIPTION OF THE INVENTION

The present invention concerns a vessel comprising an integrated tube-and-cap assembly, wherein one or more tube(s) is connected by way of one or more hinge strap(s) or device(s) to one or more cap(s).
Particularly, the invention concerns a vessel comprising an integrated tube-and-cap assembly, which assembly has been fabricated in one piece and wherein the tube and the cap have different light transmitting properties, since the material that either one of the tube and the cap is fabricated from comprises a colorant, which manipulates its light transmitting properties, while the other one is fabricated from a material, which lacks such colorant.
The vessel can comprise a single tube or a one-dimensional array having a plurality of sample tubes arranged in a line (microtiter “strip”) (FIG. 1 a).
The vessel comprises an assembly, wherein (as in FIG. 1) a tube portion (FIG. 1 b) comprises the following parts:

- 1 an upper surface
- 2 an opening
- 3 an upper wall portion
- 4 a lower wall portion
- 5 a bottom portion
- 6 a neck
  connected (FIG. 1 c) by way of:
- 7 a hinge strap or device
  to a cap portion (FIG. 1 c) comprising the following parts:
- 8 a cylindrically shaped portion
- 9 a closed dome-shaped or flat portion
- 10 a circumferential shoulder

The “tube” or particularly the “strip” of tubes may comprise an upper surface 1, from which the actual sample tube part protrudes downwardly, while its opening 2 remains on the upper surface 1. The tube is formed of an upper wall portion 3, which preferably has a cylindrical shape, a lower wall portion 4, which may also have a cylindrical shape or it may have a conically beveled shape, preferably a conically beveled shape, and a bottom portion 5.
The lower wall portion 4 extends from the upper wall portion 3 continuously, preferably so that the wall thickness is reduced as the distance from the upper wall portion 3 increases. Thus, the lower wall portion 4 is connected to the upper wall portion 3 at one end, which in the conically beveled alternative shape is the wider end, and to the bottom portion 5 at the other end. The bottom 5 of the tube thus closes the structure. The bottom portion 5 can be made thicker than the lower wall portion 4 to increase the strength of the structure but can also have the same thickness as the lower wall portion 4. If the vessel comprises a strip of tubes, the tubes are typically connected to neighboring tubes from the upper wall portions 3 by necks 6. In that case, each of the sample tubes in the vessel is connected from its cylindrical upper wall portion 3 to neighboring tubes by a neck 6. Thus, the necks 6 form the upper surface 1 of the strip. The sample tubes can also comprise shoulders, as described in the published patent application no. US 2005/0,084,957.
The tube is connected to a cap by way of a “hinge strap or device” 7, herein also called a “hinge-like structure”.
The term “cap” is used herein to describe any designs capable of sealing a sample tube, thereby preventing evaporation of the sample before or during storage, centrifuging, thermal cycling or any other process the tube, the cap and the sample may be used for. The cap can also be described for example as a stopper, a closure or a plug, and it is preferably manufactured from a polymeric material, such as a plastic. The cap is preferably pivotally attached to the cylindrical part of the tube and includes an essentially cylindrically shaped portion 8 that is removably insertable into said sample tube and is dimensioned to give a tight fit, thereby preventing evaporation of the tube contents. The cap further comprises a resiliently deformable closed dome-shaped or flat portion 9, which closes the structure. The dome-shaped or flat portion 9 extends away from the cylindrically shaped portion 8. The cap further comprises a circumferential shoulder 10, attached to and interposed between the mentioned cylindrically shaped portion 8 and the dome-shaped or flat portion 9, extending radially outwards from the mentioned portions 8,9.
The “light transmitting properties” of the materials used in the fabrication of the assembly of the present invention are mainly divided into four groups, i.e. opaque, optically clear, colored and reflective. These properties may be altered/manipulated for example by adding one or more colorants to the material that the tube, the cap or the hinge strap is fabricated from during the fabrication (not shown in FIG. 1).
The term “colorant”, as used herein, is meant to include pigments, nanoparticles and any other agents, which alter the light transmitting properties of the base material of the tube, for example the thermoplastic material.
The term “opaque”, as used herein to describe a material, means that the material, such as the wall of the tube, blocks or reflects light. The term “essentially opaque”, as used herein, means that the mentioned material has at least a portion, which is completely opaque, i.e. transmits very little light by reflecting or blocking most of it. The opaque portion may be a portion of the tube wall facing the directions of adjacent tubes, or other possible sources of an emitted signal, and may cover for example the cylindrical portion 3 of the tube. This is due to the cylindrical portion 3, at least in some cases, reaching above the upper edge of the well of the sample holder that the tube has been placed in. The walls of the wells prevent tube-to-tube light transmission, whereby only reflections from the walls of the wells (generally made of metal) may affect the accuracy of the results in the lower parts of the tube.
The term “optically clear”, as used herein to describe a material, means that the material, such as the cap, transmits light (being transparent or translucent). The term “essentially optically clear”, as used herein, means that the mentioned material contains at least a portion, which is optically clear, i.e. transmits light. The optically clear portion may be a portion of the cap covering at least a surface large enough for an emitted signal to pass, i.e. the portion may consist of only an optic window. The optic window may, for example, cover only the dome-shaped or flat portion 9 of the cap. For the achievement of a reliable detection, the clear material is required to be homogenous.
The term “homogenous”, as used herein, is meant to describe the material that the vessel of the present invention is manufactured of, which material has a homogenous surface with a homogenously distributed color.
“Thermal cyclers” are instruments commonly used in molecular biology for applications such as the polymerase chain reaction (PCR) and cycle sequencing, and a wide range of instruments are commercially available. “qPCR” is a quantitative polymerase chain reaction, i.e. a modification of the polymerase chain reaction, which is used to rapidly measure the quantity of a product of the polymerase chain reaction. It is preferably done in real-time.
According to one embodiment of the present invention, the tube portion of the tube-and-cap assembly has altered light transmitting properties, making it opaque, colored or reflective. According to this embodiment, the cap portion has unaltered light transmitting properties, making it colored, but of a different color than the tube portion, or optically clear.
According to another embodiment of the present invention, the cap portion of the assembly has altered light transmitting properties, while these properties are unaltered in the tube.
These embodiments, as described herein, rely on the base material used in the fabrication of the assembly being optically clear or colored. The present invention, however, is also meant to include base materials that are opaque, frosted or reflective, whereby altering the light transmitting properties may make the material optically clear in addition to the alterations described above. The assembly of the present invention may thus comprise an opaque tube and a clear cap, a clear tube and an opaque cap, a reflective tube and a clear cap, a clear tube and a reflective cap, a tube and a cap of different colors, a frosted tube and a colored cap, a colored tube and a frosted cap, a frosted tube and an opaque cap, an opaque tube and a frosted cap, a reflective tube and a frosted cap as well as a frosted tube and a reflective cap.
According to a preferred embodiment of the present invention, the tube is essentially opaque and is connected by way of a hinge strap or device 7 to an essentially optically clear cap. Preferably, the essentially opaque part of the tube is of a uniform, homogenous, opaque color, such as black or white, giving a complete opacity and thus a uniform light detection. More preferably, the opaque part of the tube is white.
According to a particularly preferred embodiment, the cap is fabricated completely from an optically clear material. The optically clear material is chosen from materials allowing for the introduction of excitation light and for the detection of an optical signal.
According to another particularly preferred embodiment, the tube is fabricated completely from an opaque material. Manufacturing not only the upper portion but also the lower portion of the tube from an opaque material prevents not only tube-to-tube light transmission, but also successfully prevents reflection of light from the walls of the wells of a laboratory instrument, such as a thermal cycling device or other similar device, from entering the tube. Further, a frosted tube, as the one used by BIOplastics, partly prevents light from entering or re-entering the tube through the wall of the tube, but an opaque tube, that according to the definition above blocks or reflects light, gives an even more accurate and more repeatable result, since the surface of the opaque tube provides a more complete prevention of light transmission.
According to a further preferred embodiment, the tube portion is fabricated from an optically clear material, whereas the cap portion is fabricated from an opaque material.
The base material of the sample tube and the cap preferably comprises a thermoplastic material, which will withstand the conditions typical for, e.g. thermal processing of biological samples, involving heating cycles increasing the temperature up to more than 80° C. In addition, the material should exhibit good hydrophobicity and low interference with molecular biological reactions. Examples of suitable materials include various polyolefin grades, polyesters and polycarbonates. A particularly preferred material is polypropylene, preferably of a grade suitable for melt processing, e.g. by injection molding, pressure forming, vacuum forming, extrusion molding or blow molding. The polypropylene can be nucleated or non-nucleated and it can contain heat and light F stabilizers, antistatic agents, antioxidant, nanoparticles as well as fillers, such as mica, calcium carbonate, talc and wollastonite, and pigments, such as carbonate, titanium dioxide, carbon black, quinacridone, phtalocyanine blue and isoindolinone. Preferably, the pigment is either titanium dioxide, making the material white, or carbon black, making the material black. More preferably, the pigment is titanium dioxide. Other thermoplastic resins suitable for the present purposes are various high-quality polyethylene, polybutylene and poly(ethyelene terephthalate) grades.
Preferably, either the tube or the cap is fabricated from a thermoplastic material containing a colorant, such as a pigment, nanoparticles or another agent altering the light transmitting properties of the material, while the other is fabricated from a thermoplastic material lacking such a colorant. More preferably, the material of the tube contains colorant, while the material of the cap lacks colorant. Most preferably, the thermoplastic material is polypropylene and the colorant is chosen from agents, which make the material opaque.
The thickness of the tube wall is preferably about 0.002 inches to about 0.030 inches (approximately 0.05 mm to 0.76 mm), more preferably 0.002 inches to about 0.0065 inches (approximately 0.05 mm to 0.17 mm) or even more preferably 0.002 inches to about 0.009 inches (approximately 0.05 mm to 0.23 mm), the achievable thickness being dependent upon size of area and part geometry.
Particularly, the wall thickness of the upper wall portion 14 can be, for example 0.009-0.030 inches (0.23 mm to 0.76 mm). The lower wall portion 16 can be manufactured to have a uniform wall thickness of 0.0025 to 0.0065 inches (approximately 0.06-0.17 mm). The consistency of the thickness is high with the maximum deviation from the desired wall thickness usually being below 25%, even below 10%, depending on the shape of the tube and the desired wall thickness. This leads to an even heat transfer to the reagent sample. That is, the thermal contribution of the vessel diminishes as its mass becomes smaller in relation to the mass of the sample.
The thickness of the dome-shaped or flat portion 9 of the cap can be, for example 0.002-0.009 inches (0.05-0.23 mm), whereas the thickness of the cylindrical portion 8 of the cap can be, for example 0.002-0.030 inches (0.05-0.76 mm).
The tube and the cap, as mentioned above, comprise a one-piece assembly. The portions, i.e. the tube and the cap, of the assembly are preferably connected by way of a hinge strap or device, herein also called a hinge-like structure. The material of this hinge-like structure is preferably polypropylene with or without colorant.
According to a preferred embodiment of the present invention, the above-mentioned assembly comprises a single tube attached to a single cap through a hinge-like structure. The tube may have any conventional tube size, such as 0.2 ml, 0.5 ml, 0.6 ml, 1.0 ml, 1.5 ml or 2.0 ml. For use in PCR the tube is preferably dimensioned to fit 0.2 ml or 0.5 ml of liquid.
According to another preferred embodiment of the present invention, the above-mentioned tube-and-cap assembly comprises a strip of tubes, wherein each tube is attached to a single cap through a hinge-like structure. One strip preferably comprises 8 or 12 tubes. The tubes in the strip of tubes may have any conventional tube size, such as 0.2 ml, 0.5 ml, 0.6 ml, 1.0 ml, 1.5 ml or 2.0 ml.
According to another preferred embodiment of the present invention, the above-mentioned tube-and-cap assembly comprises a strip of tubes attached to a strip of caps through one or more, preferably one or two, more preferably only one, hinge-like structure. As mentioned above, one strip of tubes preferably comprises 8 or 12 tubes. Further, one strip of caps also preferably comprises 8 or 12 caps. The tubes in the strip of tubes may have any conventional tube size, such as 0.2 ml, 0.5 ml, 0.6 ml, 1.0 ml, 1.5 ml or 2.0 ml.
The sample vessel assembly of the present invention will be compatible with general laboratory equipment and analytical instrumentation that are designed to accept tubes or strips of tubes of the above-mentioned sizes. Such general lab equipment includes centrifuges, thermal cyclers, simple heaters and chillers and liquid handlers, and the analytical instrumentation includes DNA automated sequencing systems, emission and calorimetric plate readers, and PCR instruments, such as real-time, quantitative PCR instruments. The tubes should be capable of placement into unrestricted heat transfer connection with the holder/heating means of the analyzing equipment. Preferably, the sample vessel assembly is fabricated using materials making it suitable for optical or visual detection.
In a typical application, the sample vessel assembly of the present invention is used for performing a PCR process in a thermal cycler. Such cyclers comprise a sample holder, which is designed to receive the tube or the strip of tubes and to provide a thermal pathway between a heating/cooling element of the device and the sample vessels. Preferably, the assembly is used for performing a qPCR process, more preferably for performing a qPCR process using the SYBR green method or the fluorescent reporter probe method, most preferably using the fluorescent reporter probe method. Other typical applications for the assembly include centrifuging, heating, chilling, storing, sequencing and other analytical applications.
According to a preferred embodiment of the present invention, the vessel having an integrated tube and cap is fabricated by use of a two-step molding process.
A “two-step molding process”, as used in the present invention, refers to a molding technique, wherein the high pressure injection molding machine and the specific mold can accommodate the introduction of two separate resins in one molding cycle which consists of two polymer injection steps, one for each mentioned color, e.g. an opaque color and an optically clear color, a reflective color and an optically clear color, or white and black. Common plastics molding techniques include injection molding, pressure forming, vacuum forming, extrusion molding or blow molding. When manufacturing bi-component or multi-component plastic articles, such as in some cases when manufacturing toothbrushes, a two-step or multi-step molding technique can be used. When manufacturing the vessels of the present invention, it is preferred to use a two-step injection molding technique, as described above.
In injection molding, the material, such as the plastic, to be molded generally is added to the molding device in the form of pellets. If another material, such as a colorant, is to be added, it is done in the injection stage. Particularly when manufacturing thin-walled structures, the color of the molded structure may remain inhomogeneous, due to the uneven distribution of the colorant. In the present invention, to resolve this problem the plastic material and the colorant are first pre-mixed, whereby the pellets added to the molding device already contain an essentially homogeneous mixture of plastic and colorant.
In the two-step manufacturing process of the present invention, the first step is the molding of either the tube or cap component and the second step is the molding of the complementary component, e.g. cap with tube or vice versa, allowing the joining of the two different pigments in the area of the hinge strap.
The present invention also provides a method for achieving an accurate optical signal in the detection of a thermal cycling process, wherein the sample to be detected is located in a vessel of the present invention and the signal is obtained by sending excitation light through the cap of the mentioned vessel and detecting the emitted signal returning through the same mentioned cap of the mentioned vessel.
According to a preferred embodiment of the invention, tube-to-tube light transmission is prevented in the above described method by the presence of a colorant comprised in the tube material.

Claims

1. A vessel comprising an integrated tube-and-cap assembly, wherein one or more tube(s) is connected by way of one or more hinge strap(s) or device(s) to one or more cap(s), characterized in that

the assembly has been fabricated in one piece,

the tube and the cap have different light transmitting properties, since the material of either one or both of the tube and the cap comprises one or more colorants, which alter its light transmitting properties, and

the materials of the tube and the cap do not comprise the same colorant or mixture of colorants.

2. The vessel of claim 1, wherein the material of the tube comprises one or more colorants and the material of the cap lacks colorant.

3. The vessel of claim 1, wherein the material of the cap comprises one or more colorants and the material of the tube lacks colorant.

4. The vessel of claim 1, wherein the material of the tube comprises one or more colorants and the material of the cap comprises a different colorant or different mixture of colorants than the material of the tube.

5. The vessel of any of claims 1 to 4, wherein the cap is fabricated from a homogenous, optically clear material.

6. The vessel of claims 5, wherein the optically clear material comprises polypropylene.

7. The vessel of claim 1, wherein the tube is fabricated from an opaque material.

8. The vessel of claim 7, wherein the opaque material comprises a homogenous mixture of polypropylene and one or more colorants.

9. The vessel of claim 1, wherein the colorants are chosen from pigments, nanoparticles or other agents capable of altering the light transmitting properties of a material.

10. The vessel of claim 9, wherein the colorant is titanium dioxide, giving the material a white color, or carbon black, giving the material a black color.

11. The vessel of claim 10, wherein the colorant is titanium dioxide.

12. The vessel of claim 10, wherein the colorant is carbon black.

13. The vessel of claim 1, wherein the integrated tube-and-cap assembly comprises a single tube connected to a single cap by way of a hinge strap or device.

14. The vessel of claim 1, wherein the integrated tube-and-cap assembly comprises a strip of tubes, wherein each tube is connected to a single cap by way of a hinge strap or device.

15. The vessel of claim 1, wherein the integrated tube-and-cap assembly comprises a strip of tubes, wherein the tube strip is connected to a strip of caps by way of one or more hinge straps or devices.

16. Use of a vessel of claim 1 for thermal cycling, centrifuging, heating, chilling, storing, sequencing and other analytical applications.

17. Use of a vessel of claim 1 for performing a PCR process.

18. A method for achieving an accurate optical signal in the detection of a thermal cycling process, wherein the sample to be detected is located in a vessel of claim 1 and the signal is obtained by sending excitation light through the cap of the mentioned vessel and detecting the emitted signal returning through the same mentioned cap of the mentioned vessel.