WO1992020448A1

WO1992020448A1 - Microplate for containment of radioactive samples

Info

Publication number: WO1992020448A1
Application number: PCT/US1992/002183
Authority: WO
Inventors: Deryl B. Stanley
Original assignee: Packard Instrument Company, Inc.
Priority date: 1991-05-15
Filing date: 1992-03-17
Publication date: 1992-11-26

Abstract

A microplate (10) for radiometric analysis having a plurality of sample wells (12) for holding radioactive samples is manufactured from a gas barrier resin which is chemically resistant to hydrocarbon solvents. The resin is typically a rubber modified copolymer which includes at least 50 weight percent of an unsaturated nitrile component and a monomer component which is capable of being copolymerized with the nitrile component. A preferred gas barrier resin is an acrylonitrile-methyl acrylate copolymer. A white pigment, preferably titanium dioxide, is added to the resin to produce an opaque, highly reflective microplate (10).

Description

MICROPLATE FOR CONTAINMENT OF RADIOACTIVE SAMPLES

Background Of he Invention

The present invention relates generally to microplates utilized in radiometric analysis systems, and more particularly, to microplates for containment of multiple radioactive samples. The use of multiple sample microplates for containing cell cultures and the like has become common place in the field of biotechnology. In many situations, the cell cultures are labeled with radioactive isotopes such that the radioactivity of the final samples must be measured. A total of up to ninety six samples are commonly contained in a typical microplate, each of which is separately measured by a radiometric analysis system some of which utilize liquid scintillators for detecting radiation.

Each of the sample wells is provided with a scintillator that converts radiation, such as beta particles, into corresponding light pulses. As an example, a predetermined amount of a radioactive sample and a liquid scintillation cocktail is placed in a sample well before the microplate is loaded into a counting chamber of a scintillation spectrometer. Then, as the radionuclide in the sample decays, emitted beta particles energize the fluor contained within the liquid scintillation cocktail. The fluor converts the energy from the beta particles into optical events which are detected by a photomultiplier tube in the scintillation spectrometer. The scintillation spectrometer includes at least one photomultiplier tube which senses scintillation from each sample well and converts the sensed scintillation into corresponding electrical pulses. For example, the 'TopCount" scintillation spectrometer manufactured by Packard Instrument Company and described in United States Patent Application No. 07/414,678, filed September 29, 1989, and incorporated herein by reference, describes a system for measuring the radioactivity of samples using a single photomultiplier tube for sensing sample scintillation and converting them into corresponding electrical pulses. These electrical pulses are processed to discriminate between pulses attributable to sample events within the wells and pulses attributable to non-sample events such as photomultiplier tube noise. The electrical pulses attributable to sample events are supplied to a pulse analyzer which evaluates the number and energy level of the pulses attributable to each sample.

Commercially available microplates are made of polystyrene or polypropylene. Although polystyrene is a low cost plastic which is easily molded to form disposable microplates, it is not chemically resistant to solvents that are present in the liquid scintillation cocktails. Samples containing these solvents dissolve polystyrene, thus degrading the microplate and the sample. Accordingly, samples to be tested must be prepared just prior to measurement and cannot be stored to later reverify results because of the dissolution of the polystyrene microplate and resultant sample degradation. The polypropylene microplates, while being chemically resistant, are prone to optical crosstalk which is an interference between adjacent samples during sample measurement. Crosstalk results because the translucency of the microplate allows the optical events in adjacent samples to be sensed by the photomultiplier tube when it is measuring a sample, so as to artificially increase the counts per minute of that sample.

Another disadvantage of the conventional polystyrene microplate is that it can only be sealed with a film that is bonded to the microplate by an adhesive which degrades when exposed to fumes from the solvents present in a scintillation cocktail. If the tray is covered with a hard, molded cover instead of a film, it cannot be used in the TopCount scintillation spectrometer. Crosstalk and efficiency losses would result because the photomultiplier tubes would be positioned too far away from the sample. Present radiometric analysis systems are awkward to handle because they require the sample to be enclosed in a sealed bag. In radiometric analysis systems, there has been a need for low cost disposable microplates which may be used to contain and store radioactive samples containing liquid scintillators. An opaque, dimensionally stable microplate which is chemically resistant to the solvents within the scintillator and exhibits high radioactive counting efficiency while imiώmzing crosstalk between adjacent sample wells is required to assure accurate measurement of sample events. Summary Of The __.¥?_ "

It is a primary object of the present invention to provide an improved microplate which is capable of containing and storing a plurality of radioactive samples in a plurality of sample wells in which such samples are normally cultured or otherwise prepared. In this connection, a related object of this invention is to provide such an improved microplate which is chemically resistant to the solvents included in the radioactive samples.

Another important object of this invention is to provide a cost effective, disposable, dimensionally stable improved microplate which is substantially impervious to oxygen so as to minimize sample evaporation.

It is another object of this invention is to provide such an improved microplate which is heat sealable with a polymeric film to prevent contamination spills within a scintillator spectrometer and to enable storage of radioactive samples. Yet another object of tbis invention to provide such an improved microplate which is light colored to increase reflectivity so as to have a high count efficiency, and which is opaque to minimize crosstalk between radioactive samples in adjacent sample wells.

Other objects and advantages of the invention will be apparent from the following detailed description and the accompanying drawings.

In accordance with the present invention the foregoing objectives are realized by providing a microplate having a plurality of sample wells for holding radioactive samples for radiometric analysis. The plate is manufactured from a gas barrier resin which is chemically resistant to hydrocarbon solvents. The resin is typically a rubber modified copolymer which includes at least 50 weight percent of an unsaturated nitrile component, and a monomer component which is capable of being copolymerized with the nitrile component. A preferred gas barrier resin is an acrylonitriie-methyl acrylate copolymer. A white pigment, preferably titanium dioxide, is added to the resin to produce an opaque microplate. In a preferred embodiment, the microplate as above described is covered with an optically clear fil which seals each of the upwardly open sample wells of the microplate to prevent the contamination of samples and the radiometric analysis system. The film is also made of a gas barrier resin which is chemically resistant to hydrocarbon solvents. Brief Description Of The Drawings

In the drawings: FIG. 1 is a partial top plan view of a microplate embodying the present invention; and

FIG. 2 is a partial cross sectional view taken along the line 1-1 of FIG. 1. While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and will herein be described in detail. It should be understood, however, that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appended claims. Detailed Description Of The Preferred Embodiment

Turning now to the drawings and referring specifically to FIG. 1, there is shown a microplate 10 which is made of a gas barrier resin and defines a plurality of sample wells 12 which each hold a radioactive sample (not shown). As illustrated in FIG. 2, each of the plurality of wells 12 includes a bottom wall 14 which defines the bottom of the sample well. The bottom wall 14 is continuously connected to a cylindrical side wall 16 which defines the sides of the sample well 12. At the top of each cylindrical wall 16, a rim 18 formed around the perimeter of the wall 16 provides a raised surface 20 above the sample well 12 to provide a sacrificial sealing surface. The plurality of sample wells 12 are arranged in rows and columns to ensure sample identification as will be described below. Below the rims 18 of the plurality of sample wells 12, a surface 22 is formed between the plurality of sample wells 12 and around the row and column arrangement referred to above.

Each of the plurality of sample wells 12 is connected to vertically and horizontally adjacent sample wells by a connector 24 formed below the surface 22 to add rigidity to the microplate 10. The surface 22 has side walls 26 for supporting the plurality of sample wells 12 and the surface 22. The microplate rests on a base 28 that extends beyond the side walls 26. In order to provide a rigid microplate, a plurality of enforcing elements 30 extend from some of the connectors 20 to the base 28.

In order to ensure identification of the radioactive samples within the plurality of sample wells 12, identification marks in association with each of the wells may be formed on the surface 22. A conventional form of identification marks would include alphabetical letters to indicate rows and numbers to designate columns so that each sample well would have an individual numerical and alphabetical designation as shown in FIG. 1. In accordance with a further feature of the invention illustrated in FIG. 2, a transparent cover film 32 is sealed to each rim 18 of the plurality of sample wells 12 to prevent contamination spills within the radiometric analysis system and to provide for storage of the radioactive samples. In order to ensure heat sealability, the cover film 32 is also constructed of a transparent gas barrier resin that is chemically resistant to hydrocarbon solvents. The film 32 is sufficiently thin to prevent transmission of light to the photometer beyond the individual sample well that is being measured. Preferably, a 1.3 mil thin film is die cut to size and is placed over the surface 22 of the microplate 10. The film 32 is sealed to the surface 20 of each rim 18 by exposing the surface 22 to heat for several seconds. This exposure individually heat seals each of the plurality of sample wells 12. The extra film surrounding the column and row arrangement of the sample wells 12 is torn off after the film is heat sealed in order to prevent the film from extending over the edges of the outermost sample wells so as to hinder automated handling of the microplates. In order to provide a dimensionally stable, disposable, heat sealable microplate which prevents crosstalk between adjacent radioactive samples, the microplate is integrally formed from a gas barrier resin which is chemically resistant to hydrocarbon solvents. The gas barrier resin is a copolymer containing at least 50 weight percent of an unsaturated nitrile monomer and a second monomer which is capable of being copolymerized with the unsaturated nitrile monomer. These high nitrile resins have excellent transparency, rigidity, processability and gas barrier resistance to oxygen, carbon dioxide and other gases. The resins are highly chemically resistant to solvents such as benzene, toluene, xylene, 1, 2, 4-trimethylbenzene (pseudocumene), alkobenzenes, diisopropyl napthalene, phenylxylylethane (PxE), heptane and ethyl acetate. One or more of the aforementioned solvents are usually present in the liquid scintillation cocktails contained within the plurality of sample wells. Although these solvents dissolve the conventional polystyrene microtiter plates, they do not affect the composition of the microplates of the present invention. Moreover, the resins easily bond to thermoplastic films by ultrasonic, heat or impulse techniques. Since the solvent fumes of the radioactive samples may attack adhesives, the film is preferably heat sealed to the microplate. Additionally, these resins form a microplate that does not deteriorate when exposed to ultraviolet light during storage.

Preferably, the unsaturated nitrile monomer of the gas barrier resin is selected from the group consisting of acrylonitrile and methacrylonitrile. The monomer capable of being copolymerized with the unsaturated nitrile is an ethylenically unsaturated copolymerizable monomer selected from the group consisting of alkyl aαylates, alkyl methacrylates, acrylic acid or methacrylic acid. According to one embodiment of the invention, the gas barrier resin of which the microplate and cover film are constructed is a rubber modified acrylonitrile- methylacrylate copolymer containing about 75 weight percent acrylonitrile and about 25 weight percent methylacrylate. Such a rubber modified copolymer resin is commercially available under the trademark Barex 210-1® resin manufactured by British Petroleum Chemicals Corporation.

The gas barrier resin of the present invention includes from 5 to 95 weight percent, preferably, from about 60 to about 90 weight percent unsaturated nitrile monomer and from 5 to 95 weight percent, preferably, from about 10 to about 40 weight percent copolymerizable monomer. If the unsaturated nitrile is present in an amount less than 5 percent, the processability of the gas barrier resin is inadequate. If more than 95 weight percent of unsaturated nitrile is present, the chemical resistance of the microplate is adversely effected.

The microplates of the present invention are opaque to rαiαimize crosstalk and are light in color so as to be highly reflective in order to ensure high counting efficiency with respect to the radioactive samples. To form an opaque light colored microplate, a pigment having a high Albedo, preferably white, is added to the gas barrier resin in an amount from about 2 to about 20 weight percent. If a greater amount of pigment is added, the resin is too viscous for injection molding. The white pigment is selected from the group consisting of titanium dioxide, zinc oxide, zinc sulfide and thithopone. According to one embodiment of the invention, from about 4 to about 17 percent titanium dioxide is added to the gas barrier resin because of the enhanced hiding power of titanium dioxide. For example, titanium dioxide hides the natural amber color of the pH washed Barex-210-I^® resin to provide greater microplate opacity. Moreover, titanium dioxide is more chemically resistant to the scintillation cocktail solvents described above.

The gas barrier resins of the present invention are integrally formed into microtiter plates by conventional injection molding techniques. For example, Barex 210-1® is fed into a hopper in bead form, is melted and is driven into a mold in a semi-viscous state while under pressure. The resin disperses before it hardens to form a microtiter plate.

Example The microplates of the present invention were constructed by injection molding pH washed Barex 210-I^W resin containing from 13 to 17 weight percent titanium dioxide colorant. A cocktail formulation known as Microscint manufactured by Packard Instrument Company, was placed in each sample well. The samples were tested to determine count efficiency with a TopCount scintillation spectrometer. The average counting efficiency for each microplate is listed below according to the percentage of colorant in the microplate.

Microfluor microplates constructed of polystyrene as manufactured by Dynatech were also tested. These microplates dissolved in the presence of the

M roscint scintillation cocktail so that counting efficiency could not be determined.

The foregoing description is not limited to the specific embodiment herein described, but rather by the scope of the claims which are appended hereto. For example, although the invention has been described with reference to cylindrical sample wells, the sample wells may be U-shaped or slightly flared toward the rim.

Films of different thicknesses than those particularly described may be used to seal the sample wells as long as the light transmitted by the photometer through the film does not extend beyond the individual sample well being measured so as to cause light piping.

Claims

CLAIMS:

1. A microplate having a plurality of sample wells for holding radioactive samples for radiometric analysis, wherein the microplate is integrally formed from a gas barrier resin which is chemically resistant to hydrocarbon solvents.

2. The microplate of claim 1 wherein said resin is a rubber modified copolymer comprised of at least 50 wt.% of an unsaturated nitrile component and a monomer component which is capable of being copolymerized with said unsaturated nitrile component.

3. The microplate of claim 2 wherein the monomer component is an ethylenically unsaturated monomer selected from the group consisting of alkyl acrylates, alkyl methacrylates, acrylic acid and methacrylic acid.

4. The microplate of claim 2 wherein the unsaturated nitrile component is acrylonitrile.

5. The microplate of claim 2 wherein said resin is an acrylonitrile- methylacrylate copolymer.

6. The microplate of claim 5 wherein said acrylonitrile-methylacrylate copolymer contains about 75 wt.% acrylonitrile and about 25 wt.% methylacrylate.

7. The microplate of claim 2 wherein said microplate includes from about 2 to about 20 wt.% white pigment for opacity and reflectivity.

8. The microplate of claim 7 wherein said pigment is selected from the group consisting of titanium dioxide, zinc oxide, zinc sulfide and thithopone.

9. A microplate arrangement for storing radioactive samples, wherein the arrangement comprises: an integrally formed microplate comprised of a base having a plurality of upwardly open sample wells for receiving samples; and a film which seals each of the plurality of upwardly open sample wells to prevent the contamination of samples, wherein said microplate and said film are constructed from a gas barrier resin which is chemically resistant to hydrocarbon solvents.

10. The microplate arrangement of claim 9 wherein the film is transparent for transmission of light into each sample well, and the microplate is opaque to provide reflectivity for high count efficiency.

11. The microplate arrangement of claim 9 wherein the film is heat sealed to the perimeter of each of the plurality of upwardly open wells.

12. The microplate apparatus of claim 9 wherein said resin is a rubber modified copolymer comprised of at least 50 wt% of an unsaturated nitrile component and a monomer component which is capable of being copolymerized with said nitrile component.

13. The microplate arrangement of claim 12 wherein the monomer component is an ethylenically unsaturated monomer selected from the group consisting of alkyl acrylates, alkyl methacrylates, acrylic acid and methacrylic acid.

14. The microplate arrangement of claim 12 wherein the unsaturated nitrile component is acrylonitrile.

15. The microplate arrangement of claim 12 wherein said resin is an acrylonitrile-methylacrylate copolymer.

16. The microplate arrangement of claim 15 wherein said acrylonitrile- methylacrylate copolymer contains about 75 wt.% acrylonitrile and about 25 wt.% methylacrylate.

17. The microplate arrangement of claim 12 wherein said plate includes from about 4 to about 17 wt.% white pigment for opacity and reflectivity.

18. The microplate arrangement of claim 17 wherein said pigment is selected from the group consisting of titanium dioxide, zinc oxide, zinc sulfide and tbithopone.

19. The microplate arrangement of claim 18 wherein said pigment is titanium dioxide.