Patent Grant
7354774
1. Field of the Invention
The present invention relates to a device and method for chemical processing of a biological sample, and more particularly to a self-aliquoting sample dispensing assembly. The dispensing assembly, which comprises a dispensing unit and a storage unit, has a plurality of receptacles and is capable of dispensing a sample substantially simultaneously into each of the plurality of receptacles. The dispensing assembly is well suited for dispensing samples for subsequent high throughput screening, and is particularly useful for dispensing, storing and transporting biological samples for subsequent clinical analysis.
2. Description of Relevant Art
Presently, across a broad range of technology-based business sectors, including the chemical, bioscience, biomedical, and pharmaceutical industries, it has become increasingly desirable to develop capabilities for rapidly and reliably carrying out chemical and biochemical reactions in large numbers using small quantities of samples and reagents. Carrying out a massive screening program manually, for example, can be exceedingly time-consuming, and may be entirely impracticable where only a very small quantity of a key sample or component of the analysis is available, or where a component is very costly.
In order to perform this function effectively, systems and methods have been developed for accurate and rapid dispensing of liquid samples and/or reagents, for example into multi-well plates. Typical multi-well plates contain 96, 192, 384, or 1536 receptacles which must be filled with a predetermined amount of a liquid sample.
Conventional pipettes are known which can accurately dispense a known quantity of liquid sample into a receptacle or other container. Manual pipettes have the obvious limitation of requiring sequential operation which is time consuming and inefficient. More automated devices, such as multi-channel pipetters are commercially available and represent an improvement over manually operated pipettes. In one example, a 96 channel pipetting device using positive displacement plungers in corresponding cylinders to draw in and expel liquid via a sampling/mltering step is known. Devices of this type are often complicated mechanisms and can be prone to problems in regulating the amount of liquid dispensed, controlling splashing, maintaining sterility, and the like.
In clinical diagnostic settings, it has often been necessary to collect biological samples such as whole blood, red blood cell concentrates, platelet concentrates, leukocyte concentrates, bone marrow apirates, plasma, serum, cerebral spinal fluid, feces, urine, cultured cells, saliva, oral secretions, nasal secretions and the like in various containers or tubes for subsequent testing and analysis. Typically, the samples must then be transported to a different location, such as a laboratory, where personnel conduct specific tests on the samples. Specific tests include experiments such as, for example, protein quantification, 2-D gel plotting of proteins, drug development, Western blotting, reporter gene analysis, immunoprecipitations, epitope tagging, specific protein activity assays, etc.
It is very desirable to rapidly detect and quantify one or more molecular structures in a sample. The molecular structures typically comprise ligands, such as antigens and antibodies. Ligands are molecules that are recognized by a particular receptor. Ligands may include, without limitation, agonists and antagonists for cell membrane receptors, toxins, venoms, oligosaccharides, proteins, bacteria and monoclonal antibodies. For example, cell and antibody detection is important in numerous disease diagnostics. In recent years there has been an increase in interest in the field of biological, medical and pharmacological science in the study of nucleic acids obtained from biological samples. For example, DNA or RNA sequence analysis is very useful in genetic and infectious disease diagnosis, toxicology testing, genetic research, agriculture and pharmaceutical development. In particular, genomic DNA (gDNA) isolated from human whole blood can provide extensive information on the genetic origin and function of cells. This information may be used in clinical practice, e.g., in predisposition testing, HLA typing, identity testing, analysis of hereditary diseases and oncology. The gDNA is analyzed via many molecular diagnostic downstream procedures (e.g., micro-array analysis, quantitative PCR, real time PCR, Southern Blot analysis, etc).
In particular, nucleic acid-based analyses often require sequence identification and/or analysis such as in vitro diagnostic assays, high throughput screening of natural products for biological activity, and rapid screening of perishable items such as donated blood or tissues, for a wide array of pathogens. There has been a convergence of progress in chemistry and biology. Among the important advances resulting from this convergence is the development of methods for identifying molecular diversity and for detecting and quantifying biological or chemical material. This advance has been facilitated by fundamental developments in chemistry, including the development of highly sensitive analytical methods, solid-state chemical synthesis, and sensitive and specific biological assay systems. For example, Sanger Sequencing, blotting techniques, microplate assays, polymerase chain reaction, hybridization reactions, immunoassays, combinatorial libraries, proteomics and the like.
Traditional medical lab tests for biological samples require that the sample be obtained, transferred to a collection tube and then sent to a lab for analysis. Clinical analysis often requires the use of systems for metering, dispensing and mixing reagents with sample fluids. The sample fluids may include, for example, tissue samples, blood samples, urine samples or minute quantities of deoxy ribonucleic acid (hereinafter “DNA”) sequences in a buffer fluid. Both manual and automated systems have been available for aliquoting the fluid samples and assaying the samples with one ore more reagents. Manual systems have historically included the glass capillary pipette, the micropipette, precision syringes and weighing equipment. A variety of biological assays have been and continue to be conducted with manual equipment of the type described.
Typical methodologies, which require that the sample be distributed in a serial manner, are cumbersome. There remains a need for an apparatus and method capable of distributing a fluid sample to multiple containers evenly by a single process at the same time.
Thus, there is a present need for an automated system capable of dispensing a predetermined amount of liquid into multi-well plates and the like which is accurate, quick, and if required, preserves the sterility of the sample being dispensed for processing and/or storage.
The present invention relates to a sample dispensing unit and method for processing a sample, such as a biological sample, and more particularly to a self-aliquoting sample dispensing assembly having a storage unit and a dispensing unit arranged at a top thereof. The storage unit includes multiple receptacles into which a sample may be dispensed and a sealing member for providing an air tight seal between receptacles of the storage unit and ambient air. The sealing member includes an access member for providing a sample to be dispensed from the dispensing unit to the storage unit. The device and method function using temperature change to generate a pressure differential in the receptacles which pulls a predetermined amount of sample into each receptacle. The vacuum or a combination of vacuum and gravity enable the assembly to dispense a sample substantially equally among receptacles.
The present invention applies not only to certain fields within the chemical industry such as biotechnology, biochemistry and the like, but is also suitable for carrying out research in biological chemistry, inclusive of microbiology, or various kinds of chemical reaction tests such as a clinical diagnosis.
A self-aliquoting dispensing unit for use with a multi-receptacle storage unit is provided including: a lower plate having a plurality of access ports therethrough wherein at least one of the access ports is in fluid communication with a microtube; an upper plate releasably attached to the lower plate with the upper plate including a sample port for supplying a sample to the dispensing unit; and a sealing member for forming a reversible air tight connection between the storage unit and the upper plate.
Also provided is an assembly for dispensing a sample to a plurality of receptacles. The assembly includes a self-aliquoting dispensing unit and a storage unit in fluid communication with the dispensing unit. The dispensing unit includes a lower plate having a plurality of access ports therethrough, wherein at least one of the access ports is in fluid communication with a microtube, an upper plate releasably attached to the lower plate, the upper plate including a sample port for supplying a sample to the dispensing unit, and a sealing member for forming a reversible air tight connection between the storage unit and the upper plate.
Additionally, a method of dispensing a sample into a storage unit having a plurality of receptacles is provided including the steps of: adding a sample to a dispensing unit, attaching the storage unit to the dispensing unit to form an assembly, and creating a temperature generated vaccum in the receptacles of the storage unit to dispense an aliquot of the sample into each of the receptacles. The dispensing unit includes a lower plate having a plurality of access ports therethrough, wherein at least one of the access ports is in fluid communication with a microtube, an upper plate releasably attached to the lower plate, the upper plate including a sample port for supplying a sample to the dispensing unit, and a sealing member for forming a reversible air tight connection between the storage unit and the upper plate.
A kit for processing a sample is provided, including a dispensing assembly and reagents for processing a sample. The dispensing assembly includes a lower plate having a plurality of access ports therethrough, wherein at least one of the access ports is in fluid communication with a microtube; an upper plate releasably attached to the lower plate, the upper plate including a sample port for supplying a sample to the dispensing unit; and a sealing member for forming a reversible air tight connection between the storage unit and the upper plate.
The dispensing unit of the invention is adapted to allow chemical reaction in a receptacle so that a reaction test, for example, may be made in a simple and efficient manner.
It is an advantage of the present invention, that a dispensing unit for multi-receptacle storage units is provided which is disposable and can be used in single-use applications and then discarded.
It is a further advantage of the present invention that a dispensing unit is provided which may be maintained in a sterile condition during use.
An additional advantage of the present invention is the ability to fill a large number of receptacles with a predetermined amount of sample substantially simultaneously in one operation.
Yet a further advantage of the present invention is the ability to refrigerate or cryogenically freeze the storage unit for long-term use. In addition, the receptacles can be permanently affixed to the storage unit, removably attached to the storage unit or held in place by the storage unit and easily removed for further use. Most notably, is that the user can remove an individual receptacle for experimentation without disturbing the fluid sample in other receptacles.
Yet a further advantage of the present invention is that a dispensing unit having high precision and small volume fluid processing capability that can precisely aliquot small volumes of a sample fluid is provided.
Yet a further advantage of the invention is that a dispensing unit, which can mix small aliquots of sample fluid with various discreet reagents is provided.
It is yet a further advantage of the present invention to provide a dispensing unit and method for mixing a sample with reagent, which provides a uniform mixing concentration, and has high reaction and mixing efficiency.
The substance of interest or sample being tested and/or evaluated with the method and apparatus of the present invention may include small or large molecules such as drugs, potential drug candidates, metabolites, pesticides, pollutants, and the like.
The substance of interest may be cells or cellular components or fragments such as polypeptides and proteins, polysaccharides, nucleic acids, and combinations thereof. Among nucleic acids, for example, are DNA, cDNA, gDNA, RNA, MRNA, tRNA, and combinations thereof.
Furthermore, the substance of interest may be a specific binding pair (sbp) and may be a ligand, which is monovalent (monoepitopic) or polyvalent (polyepitopic), synthetic or natural, antigenic or haptenic, and is a single compound or plurality of compounds which share at least one common epitopic or determinant site.
In addition, a cell bearing a blood group antigen such as A, B, D, etc., or an HLA antigen, cell membrane receptors may be a substance of interest.
With the foregoing and additional features in mind, this invention will now be described in more detail, and other benefits and advantages thereof will be apparent from the following detailed description when taken in conjunction with the accompanying drawings, in which like elements are identically numbered throughout the several views.
The present invention provides a sample dispensing unit and method, which uses a temperature differential generated vacuum to draw a sample from a dispensing unit into multiple receptacles held in a storage unit. The vacuum or a combination of vacuum and gravity enable the assembly to dispense a sample substantially equally among receptacles.
The apparatus and method of the invention, which permit performance of chemical reactions on many samples each in a small quantity, is particularly useful in clinical diagnosis.
Fluid biological samples and other substances in solution are often stored by freezing. A sample of the frozen fluid will remain stable for extended periods as long as it is kept in the frozen state. Frequently these fluids are collected in relatively large quantities, (“collected samples”), and are used in smaller quantities, (“specimens”), over an extended period of time. When a specimen is needed, it often requires thawing the entire collected sample to obtain the specimen currently needed, and then refreezing the remainder of the collected sample. However, frequent freezing and thawing cycles are almost always detrimental to the unstable ingredients in the collected sample. Further, when, for example, the blood of a patient is used as a sample, as tests on a given volume of blood have to be made for many items, the volume of blood to be used for one item is gradually reduced. However, the aforementioned apparatus, which contains multiple receptacles each having an equal amount of a biological sample, has an advantage in that it permits each receptacle to be used as needed.
In one aspect of the invention, it is possible to store samples in multiple small individual receptacles, permitting the thawing of individual receptacles without having to thaw and refreeze the entire collected sample.
The present invention is directed to a sample dispensing unit and the method for its use. A partial vacuum is created to move the liquid sample, typically blood, into the receptacles to permit one or more analytes or characteristics of the sample to be measured, typically optically or electrochemically, or by other conventional means. The dispensing unit and method for using same permits component measurements using a small liquid sample volume, allows accurate control over the proportion of the liquid sample to reagent, provides for simplicity of use, and accommodates disposability thereof.
A primary advantage of the invention is its simplicity. It is simple in construction and thus is relatively inexpensive to produce.
Additionally, the proportion of any reagent to the liquid sample size can be accurately controlled for subsequent accurate and consistent measurement.
The sample dispensing assembly and method of the invention are especially well suited for dispensing biological samples for subsequent high throughout screening such as proteomics analysis.
Referring now to
The storage plate 8 is substantially perpendicular to the support member 10 and may include 96 orifices 12 in an 8 by 12 array sized to fit 96 receptacles 16 therein. Toward each corner of the storage plate 8 are securement apertures 14 for connecting the storage unit 6 to the dispensing unit 4.
In
It is also possible for the receptacles 16, storage plate 8 and support member 10 to be made integral. Referring to
It is to be understood that although an 8×12 array is shown, any number of receptacles of any size and configuration may be used. For example, the storage plate may be a 2×6 array, or other arrangement. In a desirable aspect of the invention, the storage unit conforms to Society for Biomolecular Screening (SBS) standards for microplates and the dispensing unit according to the invention is sized to be compatible with. As a result, it will be possible to use the dispensing unit of the invention with conventional microplates which also conform to these specifications.
The dispensing unit, generally referred to by reference number 4, includes a lower plate 26 and an upper plate or lid 28. The dispensing unit 4 includes a sealing member 24, including receptacle caps, for providing an air tight seal between the air in each of the receptacles and ambient air. In this case the combination of the plates 26 and 28 and the receptacle caps 16 serve as the sealing member.
The lower plate 26 is substantially planar with a perimetric geometry substantially corresponding to that of the storage plate 8. The lower plate 26 includes a plurality of access ports 30 therethrough, wherein each access port is associated with a receptacle into which a sample will be dispensed. Each access port 30 is in fluid communication with a microtube 32 extending downward toward a base of the receptacle for dispensing the sample thereto. The lower plate 26 is provided with a plurality of securement apertures 34 therethrough for connecting the dispensing unit 4 to the storage unit 6.
The shape of the microtubes 32 is not critical, although cylindrical is preferred. The ends of the microtubes may be sharp or blunt depending on their ability to pierce the particular material selected for use in the caps. The diameter of the microtubes is not critical. The diameter of the microtubes should be small enough so that the surface tension created by the sample is sufficient to resist sample from flowing through the microtubes and into the receptacles before creation of a temperature differential induced vacuum. The microtubes should be large enough so that a liquid will not take an extended period of time to fill the receptacles.
The upper plate or lid 28 includes a securement member for attaching the lid 28 to the lower plate 26 and/or the storage unit 6. In
The lid 28 includes a sample port 40 therethrough for supplying the sample to be dispensed. A plug 42 is provided on a top side of the lid 28 for closing the sample port 40 when it is not in use. A vent hole 44 creates a channel through the lid for allowing pressure to equilibrate between pressure in the receptacles and pressure external to the assembly 2 of the dispensing unit 4 and the storage unit 6. The vent hole may be provided with a vent hole plug 46. The plugs may be made of any suitable elastomeric material, such as natural rubber elastomers, synthetic thermoplastics, and thermoplastic elastomeric materials.
Referring now to
In one aspect of the invention the dispensing unit 4 and the storage unit 6 are supplied separately. In this case, it is possible for a user to pre-treat the receptacles 16 as required before connecting the units to form the assembly 2. For example, it will be possible to add a protease inhibitor into the receptacles 16 prior to adding a blood sample so as to inhibit degradation of the sample. Additives including cationic compounds, detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, quaternary amines, and mixtures thereof, also may be provided in the receptacles prior to addition of sample. Chemical agents can be included to permeabilize or lyse cells in the biological sample. Suitable additives include, but are not limited to, phenol, phenol/chloroform mixtures, alcohols, aldehydes, ketones, organic acids, salts of organic acids, alkali metal salts of halides, additional organic chelating agents, fluorescent dyes, antibodies, binding agents, anticoagulants such as sodium citrate, heparin, and the like, and any other reagent or combination of reagents normally used to treat biological samples for analysis.
In a further aspect of the invention, at least one of the lower plate 26 and the storage plate 8 also includes a separating member 48 for dividing the assembly into a plurality of sections that are air tight with respect to one another. In this case, there will be additional access ports 42 for supplying the sample for each of the sections. For example, it is possible for the unit to be divided into four air and liquid tight sections as shown in
Furthermore, it is possible to use the invention to dispense sample into less than the entire number of receptacles. For example, it is possible to remove some of the receptacles so that their associated microtubes are exposed to the ambient air. In this case, the sample will not be drawn out of the microtubes because the surface tension of the sample will avoid liquid from freely flowing out of the microtube and, without the air tight attachment of an associated receptacle, there will be no pressure differential for drawing out the sample. Alternatively, fewer microtubes than receptacles may be provided. In this case as well, air tight attachment is prevented and no pressure differential will exist for drawing out the sample.
Under normal use conditions, the dispensing unit will remain level during the distribution process. For example, when used on a bench top or in a refrigerator, each of the microtubes will remain on a plane such that they are even or level with one another. However, in the event the assembly is not retained in a level position during the distribution process, it would be advantageous for the dispensing unit to resist uneven distribution of sample across the surface of the lower plate. Therefore, in a desirable aspect of the invention, the distribution unit 4 includes a member for evenly distributing the sample across the surface of the lower plate 26 before the sample is dispensed.
Referring now to
Referring again to
The upper surface 60 includes a plurality of distribution cells 68 which comprise a series of hollow channels. The number of distribution cells 68 is not critical so long as they are distributed in a regular or semi-regular pattern uniformly or semi-uniformly across the portion of the distribution plate 58 inside the trough 66. In addition, a plurality of reinforcing elements 70 are arranged between the cells 68. There is no particular limitation to the number or arrangement of the reinforcing elements 70, so long as the cells 68 are reinforced so as to maintain their position with respect to one another. Once the sample is diverted to the distribution port 64, it is then distributed evenly across the lower surface 62 of the distribution plate 58.
Referring now to
In operation, the sample is drawn across the lower surface 62 by capillary action. Once the sample is drawn across the lower surface 62, it then has access to the distribution cells 68. By an equilibrating process, each of the cells 68 fill to approximately the same level. At this point, the sample is evenly distributed across the lower surface 62 of the distribution plate 58 by virtue of having been drawn up into the cells 68. In addition, the sample is distributed across a top surface of the lower plate 26 of the dispensing unit 4 and is ready to be dispensed into the receptacles 16.
Referring now to
In this aspect, rather than each well 16a being fitted with an individual cap, the sealing function is performed by a film 24a septum. The film 24a is a substantially planar sheet made of elastomeric material which when arranged between the microtiter plate 6a and the dispensing unit 4 forms a gas tight seal over the openings 52 of the wells 16a. The film 24a is pierced by the microtubes 32 when assembled with the storage unit, in this case a microtiter plate 6a. As described previously, sample is dispensed into the wells when the temperature induced vacuum is generated.
Furthermore, in this aspect of the invention, the securement aperture for connecting the microliter plate to the dispensing unit may comprise a lip 54 arranged on a periphery of the lower plate 26 of the dispensing unit 4 to form an air tight friction fit with an edge 56 of the microtiter plate 6a. The lip 54 in combination with the film 24a forms an airtight connection between the dispensing unit 2 and the microtiter plate 6a. Optionally, an adhesive is applied to an inside of the lip 54 to further secure the dispensing unit 4 to the microtiter plate 6a.
Referring now to
Optionally, the upper plate 28 may be used as the lid. When the embodiment using a gasket 76 to perform the sealing function is used, as shown in
Once removed, the dispensing unit can be disposed of in accordance with legal requirements. If the sample contains biohazard materials, such as blood, then the ends of the microtubes may be guarded so as to avoid contact with any residual sample or sharp ends of microtubes using any suitable means. Referring now to
The storage unit and the dispensing unit are desirably pre-assembled for use with the dispensing unit being installed onto the storage unit so that the microtubes have pierced the caps or film. The present invention also includes, therefore, an assembly 2 including dispensing unit 4 and storage unit 6. In one aspect of the invention, the assembly is provided with a solid, liquid or combination reagent in the receptacles.
Alternatively, the storage unit and the dispensing unit are assembled by a user prior to use. In this case, it is possible for the user to assemble the dispensing unit of the present invention with a conventional storage unit or microtiter plate. Additionally, a user may pre-treat an inside of the receptacles or wells prior to dispensing a sample therein.
There are no particular limitations to the design and construction materials of the assembly according to the invention. Preferably, the dimensions of the storage unit will comply with the Society for Biomolecular Screening (SBS) standards for microplates including standard SBS-1 Footprint Dimensions and standard SBS-4 Well Positions.
Robotics based high throughput tools are now routinely used to screen libraries of compounds, for example, to identify lead molecules for their therapeutic potential. The SBS standards are intended to serve as conformed industry standards in these types of assays to facilitate compatibility of equipment used therein. Because the distribution unit can be sized to conform to the aforementioned standards, it is possible to use the present invention in conjunction with existing robotic based methods used to automate handling of samples. See, for example, U.S. Pat. No. 5,104,621 to Pfost et al. Screening methods that can be performed using the dispensing assembly and method of the present invention include those discussed in U.S. Pat. No. 5,585,277 to Bowie et al.
With respect to the storage unit 6, the vertical support members 10 and the storage plate 8 may be constructed of a stainless steel or other rigid material such as plastic. The storage plate 8 may have any number of receptacles 16, however it is typical for 12, 96, 192, 384, or 1536 receptacle units to be used in biotechnology, drug discovery, and medical technology applications such as high throughput drug discovery applications.
The receptacles 16 may be constructed of any suitable material, desirably a polymeric material. Selection of the material will be based on its compatibility with the conditions present in the particular operation to be performed with the receptacles. Such conditions can include extremes of pH, temperature, and salt concentration. Additional selection criteria include the inertness of the material to critical components of an analysis or synthesis to be performed, such as proteins, nucleic acids, and the like. If conditions of handling the receptacles are expected to involve repeated freeze/thaw cycles, then polypropylene or high density polyethylene are preferred. Desirably, a translucent material such as polystyrene or polypropylene is used to form the receptacles, in order to allow a user to confirm proper fill level or to facilitate later spectroscopic or other detection.
Furthermore, it is desirable to provide the receptacles 16 with a bar code (not shown) toward a tip thereof for ease in identifying the sample contained therein. In applications involving multiple analyses being conducted on an individual or multiple samples, it is important to be able to track the sample including the date collected, source, technician, reagents, and the like. In addition, significant events after initial collection can be tracked including number, type, date of repeat analyses, freeze/thaw cycles, transport of the sample, and the like. The bar code can be used in conjunction with available software to track and develop reports on the data collected from the samples.
The caps 24 or film 24a may be formed of any suitable elastomeric material capable of forming an air tight seal when pierced by the microtube 32. Furthermore, the gasket 76 may be made of any suitable elastomeric material capable of forming an air tight seal between the dispensing unit 4 and the receptacles 16. Desirably, the caps, film or gasket are formed of an ethylene vinyl acetate (EVA) or a silicone rubber. One commercially available product suitable for use as a cap is the pierceable Capmat M5300 (Micronic BV, Lelystad, NE).
Furthermore, there are no particular limitations to the materials used to form the dispensing unit 4. For example, the upper plate 28, distribution plate 58, and lower plate 26 and microtubes 32 may be formed of any substantially rigid material. Particularly desirable are polymeric materials such as plastics. Non limiting examples of plastics that may be used include polycarbonate, polystyrene, polytetrafluoroethylene, polyvinyl chloride, polydimethylsiloxane, and the like.
The lower plate 26 may be formed with appropriately spaced holes according to known methods. Microtubes 32 may be formed separately of a suitably rigid material such as an elastomer or the like, and be either pressed into the access ports 30 in the lower plate 26 or bonded to the access ports 30 using any suitable material, for example, an adhesive. The lid 28 may similarly be formed with any appropriate material, which can be the same or different from that used in forming the lower plate 26. Desirably, the lid 28 will be made of a translucent or transparent plastic material so that a visual confirmation of distribution of the sample across the entire surface of the lower plate 26 can be made.
The parts of the dispensing unit 4 may be fabricated using any suitable means, including conventional molding and casting techniques, extrusion sheet forming, calendaring, thermoforming, and the like. For example, with apparatus prepared from a plastic material, a silica mold master, which is negative for the lower plate, can be prepared by methods generally known in the art. A liquefied polymer may then be added to the mold to form the part.
The function of the dispensing unit relies on a practical application of the Ideal Gas Law:
PV=NRT
wherein:
In the present invention, a pressure differential within the receptacles is created between the time the assembly is filled with a sample and the time the sample is to be dispensed. At the time of filling the dispensing unit with a sample, the pressure within the receptacles is related to the ambient temperature, for instance, that present in a laboratory hood or on a laboratory bench. Once the dispensing unit is filled at this first temperature, the assembly is then exposed to a cooler temperature. By exposing the assembly to a colder temperature, for example by placement into a refrigerator or freezer, the air inside the receptacles becomes colder. In accordance with the Ideal Gas Law, since the volume of the receptacles, the number of moles of gas within the receptacles, and R are each constant, the pressure within the receptacles is reduced commensurate with the temperature differential between the air outside the freezer and the air inside the freezer. This pressure reduction produces the vacuum which then pulls the sample substantially simultaneously through the plurality of microtubes and into the receptacles.
Deriving from the Ideal Gas Law, when N is constant, the product of a first pressure and a first volume divided by a first temperature will be equal to the product of a second pressure and a second volume divided by a second temperature, as follows:
P1V1/T1=P2V2/T2
It is therefore possible to predetermine the temperature differential required to obtain a desired volume of a sample to be dispensed into each receptacle. First, the final volume of gas remaining in the receptacle when the proper volume of liquid has been dispensed must be determined. By subtraction, the total volume of the receptacle minus the desired volume of the filled receptacle, equals the volume of gas V2 that will remain in the receptacle after it has been filled to the desired volume. The pressures P1 and P2 will be equal after the sample has been dispensed, and therefore may be removed from the equation.
As a result, starting with a given known temperature T1, where the original volume of the empty receptacle is assigned V1, the temperature T2 necessary to achieve the desired end volume V2 of gas in the receptacle is determined according to the following formula:
T2=T1V2/V1
It is possible, therefore, by performing a simple calculation, to determine the temperature differential required to achieve the desired fill volume of the receptacles.
Since the pressure differential is substantially the same throughout the entire assembly, substantially the same volume of sample will be dispensed into each of the receptacles. The receptacles are filled substantially simultaneously, the rate of which is related in part to how quickly the vacuum is produced.
There are no particular limitations to the temperatures that may be used. As long as it is possible for the sample to flow through the microtube, the dispensing unit will be able to perform its function. Therefore, the range of possible and optimal temperatures will be determined by the needs of the particular samples in question. For example, certain viscous samples will become more viscous in cooler temperatures. As a result, it is advisable to use higher temperatures for creating the pressure differential when dispensing more viscous samples. In addition, temperatures below freezing (32° F.; 0° C.) can cause many liquid samples to begin to crystallize. For these susceptible samples, it is advisable to use temperatures above freezing if the time necessary to create the required pressure differential is so long as to approach the time it takes for the samples to begin to freeze. The necessary temperature restrictions will be readily apparent to those having ordinary skill in the art.
The time necessary to dispense a sample into the receptacles varies depending on the capacity of the surrounding media to change the temperature of the receptacles. For example, water is a more conductive medium for generating a temperature change than is air. As a result, for a given fill volume, it will take longer to produce the necessary pressure differential by placing the assembly from ambient air into a refrigerator at a given temperature than by placing the assembly into a water bath of the same temperature.
Any manner of generating a temperature derived pressure differential is within the contemplation of the present invention. Therefore, although lowering the sample temperature from ambient to that of a refrigerator or freezer has been contemplated, it is equally possible to heat the storage unit or the assembly to above room temperature, for example using a water bath, before addition of the sample. The assembly may then be allowed to cool to room temperature. The samples may be transferred from one elevated temperature bath to a lower temperature bath, such as an ice bath. This operation may be performed, for example, in a sterile hood.
In a method according to the invention, an assembly of a dispensing unit and a storage unit is filled with a sample to be dispensed. A pressure differential is generated by reducing the temperature of the assembly. The assembly is then allowed to equilibrate to the lower temperature and dispense a sample into the receptacles.
Additionally, the dispensing unit may be replaced after a first dispensing event to allow for sequential additions of samples into the receptacles or wells.
In one aspect of the invention, a method is provided for dispensing a sample in a high throughput assay including the steps of adding a reagent to a multi-receptacle storage unit, assembling a dispensing unit and the storage unit into an assembly, adding a sample to the dispensing unit, and creating a temperature generated vacuum in the receptacles of the storage unit to dispense an aliquot of the sample into each of the receptacles. The sample may be analyzed after the aliquots have been dispensed. Alternatively, the samples may be stored for later analysis. Desirably, the reagent is a protease inhibitor.
The dispensing assembly and methods of the invention may be used in any of the known assay methods for analyzing samples which call for multi-receptacle handling, i.e., in which microplates are used. Non-limiting examples of such analyses include Sanger sequencing, blotting techniques, microplate assays, polymerase chain reactions, hybridization reactions, immunoassays, generating combinatorial libraries and proteomics.
After use, the dispensing unit can be removed from the storage unit. The sample unit may then be used for further handling and analysis, or to store the samples, for example in a cryogenic freezer, or the like. There is no need to transfer the sample to another receptacle for analysis or storage. When the embodiment directed to a storage unit having wells instead of tubular receptacles is used, then a lid may be placed on top of the storage unit prior to storage.
Another aspect of the present invention includes a kit for processing a sample. In one desirable aspect, a kit includes a dispensing assembly as described above and reagents for processing a sample. The reagents for the kits may be supplied already dispensed in or coated on the surface of the receptacles or packaged in a separate container or containers. The reagents may each be in separate containers or various reagents can be combined in one or more containers depending on the cross-reactivity and stability of the reagents. Desirably, the reagents include a protease inhibitor.
Under appropriate circumstances one or more of the reagents in the kit can be provided as a dry powder, usually lyophilized, including excipients, which on dissolution will provide for a reagent solution having the appropriate concentration for performing a method or assay in accordance with the present invention. The kit can also include additional reagents depending on the nature of the method for which the kit is used. For example, the kit may include solid phase extraction materials including paramagnetic beads and non-magnetic particles, lysis solutions, wash and elution and running buffers, bio-molecular recognition elements including receptors, enzymes, antibodies and other specific binding pair members, labeling solutions, substrates, reporter molecules, sample purification materials including membranes, beads, and the like. Included in the kit may also be additives such as cationic compounds, detergents, chaotropic salts, ribonuclease inhibitors, chelating agents, quaternary amines, and mixtures thereof, may be provided in the receptacles prior to addition of sample. In addition, chemical agents can be included to permeabilize or lyse cells in the biological sample.
The kit may include additional additives including but not limited to phenol, phenol/chloroform mixtures, alcohols, aldehydes, ketones, organic acids, salts of organic acids, alkali metal salts of halides, additional organic chelating agents, anticoagulants such as sodium citrate, heparin, and the like, and any other reagent or combination of reagents normally used to treat biological samples for analysis.
It will be apparent that the present invention has been described herein with reference to certain preferred or exemplary embodiments. The preferred or exemplary embodiments described herein may be modified, changed, added to, or deviated from without departing from the intent, spirit and scope of the present invention.
This application claims the benefit of U.S. Provisional Patent Application No. 60/379,397, filed on May 13, 2002.
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