Chemical apparatus and process disinfecting – deodorizing – preser – Control element responsive to a sensed operating condition
Reexamination Certificate
2000-01-03
2002-05-28
Fortuna, Ana (Department: 1723)
Chemical apparatus and process disinfecting, deodorizing, preser
Control element responsive to a sensed operating condition
C210S474000, C210S454000, C210S321840, C210S321780
Reexamination Certificate
active
06395233
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to the field of methods and devices for preparing samples for chemical analysis. This invention relates more particularly to equilibrium dialysis cells and a dialysis of a unique configuration and efficiency for assaying biological samples, as well as assaying biological samples with an automated pipettor.
There are many existing assays that measure the concentration of a desired molecule or ion in solution, referred to herein to as an analyte. For example a user might want to test for the concentration of the analytes thyroxine, estradiol or testosterone in a blood sample.
There are a wide variety of chemical analytic techniques used to detect analyte, such as chromatography, chemoluminesce, radioimmunoassay (RIA), Enzyme Immunoassay, Enzyme-Linked-ImmunoSorbent-Assay (ELISA) and flow techniques. These tests typically attach a marker to the analyte and then test for the presence of the marker to determine the presence of the analyte itself.
It is difficult however to measure analyte directly from a raw sample because in many instances the analyte is found in the sample as a solution of both free analyte and bound analyte. Bound analyte is usually bound to a high-molecular weight protein, and is a type of interference. An interference will react to the assay in the same manner as will the free analyte. More generally an interference is a substance, other than the material desired to be assayed, that also responds to the chosen analytical method thereby distorting the results, or a material that can prevent the assayed material from being measured at all.
In addition to the bound analyte there are other interferences commonly found in a raw sample. The sample may contain impurities such as larger molecules or solid components which can lead to adulterated analysis results. Impurities which commonly occur in sampling are, for example, extraneous proteins found when analyzing food; filter fibers and dust particles are commonly a problem when analyzing environmental samples.
When samples of assays contain interferences the results must be arrived at by analog means. For example, when there is bound analyte acting as an interference, analog means are arrived at by correlating the apparent result with standardized models to calculate an approximation of free analyte found in a given sample. The amount of free analyte present in the solution is estimated based on a characteristic ratio of bound analyte to free analyte found in past tests using the same technique for the same analyte.
Analog testing is essentially guesswork and at best is only an estimate of the free analyte. Analog determination is further complicated and unreliable because the ratio of free analyte to bound analyte may vary depending on the disease state of the patient, medications the patient is taking, etc.
Interferences can first be separated from the raw sample to produce a more accurate direct measurement. Equilibrium dialysis can be used for purifying a raw sample to extract the analyte to be assayed. The purified free analyte is separated from the bound analyte and other impurities and then measured alone. A dialysis cell and method of analyte separation is the subject of the present invention.
A problem with the use of equilibrium dialysis is the relatively long time it takes for the dialysis reaction to take place.
Dialysis is the separation of suspended colloidal particles in solution, the retentate, from the dissolved analyte ions or molecules of small dimensions. This separation is achieved by taking advantage of their unequal rates of diffusion through the pores of a semipermeable membrane. Equilibrium dialysis takes place across a semipermeable membrane by action of osmotic pressure. A semipermeable membrane is placed between a raw sample held in one chamber and an acceptor solution, the dialysate, held in a second chamber. The dialysate is a dialysis buffer solution that is chemically compatible with a given retentate and analyte. These two chambers together with the semipermeable membrane comprise a basic dialysis cell.
The permeability of the membrane is designed such that the analytes can migrate through the semipermeable membrane but the retentate and other interferences are excluded from migrating through to the dialysate. The retentate and other interferences are typically of a larger molecular weight and the semipermeable membrane allows only those molecules of lower molecular weight to migrate. The size or weight at which the largest molecule can migrate through a given semipermeable membrane is termed the Molecular Cut-Off Weight (MWCO).
Separation by dialysis is a slow process, the rate of dialysis depending in part on the differences in particle size and diffusion rates between the analyte and the non-analyte constituents. The rate at which the dialysis occurs depends on several other factors, some of which are the ratio of the analyte molecular weight to the membrane MWCO, the surface area of the membrane mutually contacted by the sample and the dialysate, the temperature of the two solutions and the amount of diffusion of substances that must first occur within both solutions for total equilibrium to be reached.
The diffusion that must take place in order for the dialysis to proceed to final equilibrium is slowed when the ratio of the membrane surface area that contacts either the retentate or the dialysate is small compared to the volume of its respective chamber. The reaction may also be impeded by molecules of the retentate and other interferences blocking the pores of the semipermeable membrane.
The analytes migrate through the membrane into the acceptor dialysate solution until an equal concentration on both sides of the membrane is established. As the dialysis process occurs, concentration gradients on either side of the membrane limit the rate of migration of analyte. The net migration of the analyte is directed toward the dialysate chamber and takes place as long as the concentration of the analytes in the sample chamber is larger than in the dialysate. At equilibrium the concentration of the analytes in the dialysate comprises a value identical to that of the retentate sample.
Current methods and devices used for dialysis require very long dialysis times to reach equilibrium, in some instances 17 hours or more.
One type of dialysis cell utilizes reusable blocks with injection ports in the sides to inject the sample or the dialysate. Membranes are placed between the blocks and dialysate and sample are alternately injected into the reusable blocks through the ports. A series of blocks may be sandwiched together in an alternating block/membrane/block fashion.
As used herein a first dialysis solution means either the sample or the dialysate. A second dialysis solution means the sample or dialysate as well, but the one of these two solutions not selected as the first dialysis solution.
The dialysis cell of the Nelson U.S. Pat. No. 4,963,256 utilizes an outer chamber containing a first dialysis solution. There is also provided an inner chamber which containing a second dialysis solution. The end of the inner cylindrical chamber is covered by a semipermeable membrane and inserted within the outer container. Because the semipermeable membrane of the Nelson cell covers only the end of a cylindrical member however, only about 90 mm
2
of surface area of semipermeable membrane is available to mutually contact both of the dialysis solutions for dialysis.
In addition to the great amount of time it takes to allow both liquids to equilibrate, existing dialysis cells generally cannot be readily used with automated laboratory equipment such as an automatic pipettor. In most cases a dialysis cell must be individually loaded with the sample and the dialysate solution, requiring time and expensive manual labor to accomplish.
What is needed then is a dialysis cell that can take advantage of one or more of the variables in dialysis cell design to reduce the time that it takes for dialysis to reach equilibrium. What is also needed is a method t
Diamond Ronald N.
Stark William A.
Fortuna Ana
Hollrigel Greg S.
Quest Diagnostics Investments Inc.
Stout Donald E.
Stout, Uxa Buyan & Mullins, LLP
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