Chemistry: analytical and immunological testing – Including sample preparation – Volumetric liquid transfer
Reexamination Certificate
1998-06-30
2001-04-17
Ludlow, Jan (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
Volumetric liquid transfer
C422S082000, C422S105000, C436S053000, C436S054000, C436S179000
Reexamination Certificate
active
06218193
ABSTRACT:
TECHNICAL FIELD
The invention relates to methods and apparatus for precisely handling small volumes of fluids. More specifically, the invention relates to methods and apparatus for aliquoting and assaying biological fluid samples.
BACKGROUND OF THE INVENTION
Diagnostic and other biological assays often require systems for metering dispensing and mixing reagents with sample fluids. The sample fluids may include, for example, patient samples, blood samples, or minute quantities of deoxygenated rybo nucleic 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 or more reagents. Manual systems have historically included the glass capillary pipette, the micro pipette, precision syringes and weighing equipment. A variety of biological assays have been and continue to be conducted with manual equipment of the type described.
Relatively sophisticated microbiological assays including micro-enzyme linked immunosorbent sandwich assays (hereinafter “ELISA”) can be satisfactorily, if tediously performed manually. The demands of modern antibody/antigen matching, histocompatibility typing, paternity testing, etc. on a vast scale has precipitated the development of various automated assay equipment to more quickly process large numbers of patient samples with various reagents. It is apparent that in order to perform a multiplicity of assays with a single patient sample, the amount of sample must be relatively large, or a small sample must be aliquoted into smaller divisions.
Recent advances in microbiology have provided the biotechnologist with increasingly sophisticated tools for examining genetic material. Restriction enzyme digestion and polymerase chain reaction (hereafter “RED and PCR respectively”) have provided geneticists with multiple DNA segments from a single sample for subsequent assaying. All of these advances have increased the need for sample handling and processing techniques which are beyond the ability of the heretofore manual pipetting and other standard laboratory techniques. As a result, the industry has proceeded with the development of highly automated equipment which can rapidly and repeatably handle relatively small quantities of patient samples.
The undertaking of the Human Genome Project exceeds the limits of current fluid sample handling and processing technology. The Human Genome Project is an attempt to map the entire human genetic code, nucleotide by nucleotide. The PCR and RED techniques presently available will therefore produce an extremely large numbers of nucleotide segments which must be assayed in a variety of different ways. In addition, current methods for producing the nucleotide segments are extremely expensive requiring the very wise use of the resultant sample. It is currently calculated that without further advancement in the state of the art, the cost of producing sufficient samples for laboratories around the world will be prohibitive without the development of techniques for handling much smaller samples and reagent volumes.
Therefore, a need exists for a high-precision, small volume fluid processing system which can aliquot and dispense fluid samples in extremely small volumes, react the samples with small quantities of reagents, and perform all of the other steps which may be necessary in a conventional assay. The system should also preferably be relatively highly automated so that the incidence of human error is reduced.
SUMMARY OF THE INVENTION
It is therefore an object of the present invention to provide a high precision, small volume fluid processing system which can precisely aliquot small volumes of a sample fluid.
It is further object of the invention to provide a high-precision, small volume fluid processing system which can mix small aliquots of sample fluid with various discreet fluid reagents.
It is a further object of the present invention to achieve the above objects in a system which automatically aliquots the fluid sample, introduces appropriate reagents, mixes the sample and reagents, and incubates the same in preparation for gel electro phoresis.
It is yet a further object of the present invention to achieve the above objects in a system which can precisely and repeatably handle fluid volumes as small as 0.1 &mgr;l.
The invention achieves the objects, and other objects and advantages which will become apparent from the description which follows by providing a small volume fluid processing system employing at least one small volume capillary tube. A precision linear actuator connected to a computer controlled motor acts as a pneumatic piston to precisely inspire and expire one or more fluids into or out of the capillary in a predetermined sequence.
In a preferred embodiment, the fluid processing system can include a sample fluid station for containing an initial volume of sample material in a buffer solution or water, a reagent fluid dispensing device, monitoring equipment for determining the position of a fluid segment in the capillary tube and a mechanism for precisely positioning the fluid sample handling device in a reference plane with respect to the reagent fluid dispensing device in the sample fluid station. The sample fluid handling device can intake a precise volume of sample fluid from the sample fluid station, position itself adjacent to the reagent fluid dispensing device which can dispense an appropriate reagent into an open end of the capillary tube. The monitoring device can provide information to a computer or other management system to either advance or retard the precision linear actuator so as to move the fluids appropriately in the capillary tube. If two or more fluids have been received in the capillary tube, the linear actuator can be advanced and retarded with differential velocities so as to mix the fluids in the capillary tube. Very small volumes of fluids, as little as 0.1 &mgr;l can be handles with an accuracy of ±0.01 &mgr;l and similar repeatability if the precision linear actuator is driven by a computer controlled motor rotatably connected to a precision lead screw. The system described above may optionally contain a heating and cooling system for incubating the capillary tube in a controlled manner. The heating system can include a thin layer of highly resistive, transparent material on the outside of the tube which can be electrically excited so that a heat generating current flows therethrough. A fan can be used to cool the capillary tube while it is heated or afterwards to maintain a desired temperature or quickly cool the tube. The monitoring device for determining the position of a fluid segment in a capillary tube can be of the optical type including a light emitter/detector pair or array positioned in proximity to the capillary tube.
The sample fluid handling device can form a bubble or droplet of sample, reagent or other fluid on the open end of the tube having a known volume. By positioning a second capillary tube in alignment with and adjacent to the first capillary tube the droplet can be transferred from the first tube to the second tube by advancing the tubes towards one another until the droplet touches the second tube. Capillary action draws the droplet into the second tube in a repeatable manner. In this way, very small quantities of fluid can be transferred from one capillary to another. In an aliquoting method employing the processing system, the sample fluid handling device can inspirate aliquots of fluid sample separated by air gaps therebetween for a more precise dispensing of the aliquots into separate receiving capillary tubes.
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Friedman Neal A.
Kraft Ray
Meldrum Deirdre
Seubert Ronald
Barnard Delbert J.
Ludlow Jan
University of Washington
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