WO1992021434A1

WO1992021434A1 - Devices and methods for self-contained, controlled-release mixing

Info

Publication number: WO1992021434A1
Application number: PCT/US1992/004746
Authority: WO
Inventors: Mark Ronald Nowakowski
Original assignee: Biosite Diagnostics Incorporated
Priority date: 1991-06-05
Filing date: 1992-06-04
Publication date: 1992-12-10
Also published as: JP2002514963A; EP0587795A1; AU2180592A; CA2110688A1

Abstract

The present invention is devices and processes for self-contained, time-released mixing of an aqueous solution, having three components: reagent (1) capable of liberating gas in the form of effervescent bubbles without the application of heat; microporous material (2) of predetermined surface hydrophilicity comprising pores generally transverse thereto so as to permit liquid or gas to traverse therethrough when said aqueous solution is added; reaction well (3) of sufficient volume to contain said aqueous solution, said reagent (1) and said microporous material (2), said well (3) comprising said reagent (1) and said microporous material (2) positioned such that upon addition of said aqueous solution, said aqueous solution permeates said microporous material (2), contacts said reagent (1), resulting in the formation of effervescent bubbles which are released through said microporous material (2).

Description

Devices and Methods For Self-Contained. Controlled-Release Mixing

This application is a continuation in part of App. Ser. No. 711,621 filed June 5, 1991, from which priority is claimed.

Field of the Invention This invention is in the field of devices and methods for the self-contained, time-released mixing of a liquid solution. In particular, it relates to the mixing of a reagent immersed in an aqueous solution in which the aque¬ ous solution must be physically agitated to dissolve or disperse said reagent within the aqueous solution.

Background of the Invention

Effervescence has been utilized for many years to promote the mixing of a reagent within a solution. The known methods, however, have several shortcomings, all of which are overcome by the teachings of this invention. Mixing is accomplished by the liberation of gas within a solution from an element or chemical compound without the application of heat. The effervescent chemical compound is typically in the form of a tablet, which reacts with water to liberate carbon dioxide, and randomly floats throughout the solution as effervescent bubbles are liberated. As a result of random movement of the tablet, dense reagents which are to be dissolved or dispersed in the solution, remain at the bottom of the solution con- tainer and are never properly dissolved or dispersed. Accordingly, there is a need for controlling of mixing with effervescent bubbles.

The use of pure effervescent powder does not permit one to control when the liberation of effervescent bubbles is to commence. Often, the reagent to be dissolved or dispersed takes the form of a lyophilized reagent which has an associated reconstitution time. Many mixing scenarios require that the lyophilized product partially reconstitute before the effervescent mixing begins. Another important control feature when utilizing pure effervescent powder is the rate at which effervescent bubbles are liberated. Uncontrolled bubble liberation results in a foaming action which is detrimental to complete mixing of most any solution system. More recently, methods have been demonstrated which control the rate at which reagents are delivered to a solution system. In particular, European Patent Applica¬ tion number 81401738.0 (Havey et al.) describes the use of a solid organic binder carrier which is soluble or disper- sible in water and which contains a measured quantity of a water-soluble dispersible reagent whereby the protected quantity of reagent contained within the solid organic binder is released and dissolved concomitantly as the binder is dissolved or dispersed by the aqueous solution. In some situations, this method may be a satisfactory alternative for controlling the rate at which the effer¬ vescent powder interacts with water and therefore control¬ ling the initiation of both effervescent bubbling and the rate at which bubbling occurs. However, many common mix- ing scenarios require that no additional constituents, such as solid organic binders, be dissolved or dispersed into the reagent solution. Therefore, there exists a continuing need for a device and methods capable of self- contained, time-controlled mixing of solutions which does not rely on the addition of extraneous reagents which may be undesirable or even detrimental to the solution to be mixed.

Brief Description of the Drawing

The Figure is a cross-section of the device for use with the present invention. A container 3 may have an upper opening 4 defined by wall 5 and a bottom 6. The container may be composed of plastic, glass, or other suitable materials. As shown in the Figure, added to the bottom of the container 3 is a thin layer of effervescent reagent 1 and a solid, porous element 2 which is in physical proximity to the effervescent reagent 1. The diameter of the solid, porous element 2 is such that a water-tight interference fit is made with wall 5 of container 3.

The reagent 7 to be dissolved or dispersed in the aqueous liquid sample is typically added to the upper surface of the solid, porous element 2. It should be noted that generally, the pore size of element 2 will be smaller than the particle size of reagent 7 so as to mini¬ mize the loss of the reagent to the underside of element 2 through the pores of 2. It should also be noted that the porous element has been modified to exhibit a pre¬ determined desired surface hydrophilicity so as to control the initial bubble formation, size of the bubbles, and rate of bubble production. A liquid sample containing water, such as blood, serum, or urine, would then be added to container 3 by introduction through container opening 4. After the liquid sample permeates element 2, either by capillary action or by hydrostatic pressure, it contacts the effervescent reagent 1 and chemically reacts forming effervescent bubbles. As bubbles form, they begin to migrate through the pores of component 2 to the upper surface of 2, achieving a dynamic state with the movement of the liquid in the opposite direction, from the upper surface to the lower surface of component 2 through the pores of component 2. The movement of effervescent bubbles through the fluid in contact with component 2 mechanically agitates reagent 7, dissolving or dispersing reagent 7 throughout the solution. After a suitable period of time, reagent 7 will have completely dissolved or been dispersed in the liquid sample without the aid of additional outside mechanical intervention. Summary of the Invention

The present invention is devices and apparatus for mixing an aqueous solution, having three major components:

a. reagent capable of liberating gas in the form of effervescent bubbles without the application of heat; b. porous means of predetermined surface hydro¬ philicity comprising pores generally transverse in a controlled manner thereto, so as to permit liquid or gas to transverse therethrough in a controlled manner when said aqueous solution is added to said porous means; c. reaction well of sufficient volume to contain said aqueous solution, said reagent and said porous means, positioned such that upon addition of said aqueous solu¬ tion, said aqueous solution permeates said porous means, contacts said reagent, resulting in the formation of effervescent bubbles which are released through said porous means to mix said aqueous solution.

The present invention provides a device and method for the self-contained, time-controlled mixing of an aqueous solution by the controlled release of effervescent bubbles without additional other solid organic binders which can dissolve or disperse in the solution with poten¬ tial adverse effects. In general, the teachings of this invention are applicable for use in all liquid/liquid and liquid/solid solutions where it is desired to control the mixing of solid reagents by dissolution or dispersion. However, there is a particular need for such a device and method in the area of clinical chemical analysis. To illustrate the present invention, it is hereafter described as utilized in the field of clinical chemical analysis.

Detailed Description of Preferred Embodiments

The devices and apparatus of this invention employ a powdered reagent which can liberate effervescent bubbles when in contact with water. In addition, the device has a porous means, for example, a porous plastic, polyethy¬ lene or ceramic disc, an artificial membrane, a filter, including cellulose, glass fiber and cellular acetate filters and a screen including plastic and metallic screens which has pores generally transverse to its upper and lower surfaces. The surface of the porous means is modified by plasma treatment or treatment with a surfac¬ tant to result in the exhibition of a desired surface hydrophilicity. Many reagents are naturally hydrophillic, such as cellulose acetate and glass fiber. Naturally hydrophillic materials have an affinity for attracting, adsorbing or absorbing water. On the ^• other hand, naturally hydrophobic materials include polyethylene and polyproplyene. Such materials have a tendency to repel or to fail to adsorb or absorp water. The surface hydrophilicity can be selected or predetermined to exhibit desired characteristics using a variety of methods as described herein and which are known to those skilled in the art. The pores permit a liquid or gas to traverse from one surface to the other surface of the second member. The pore size may be microporous, for example in the preferred embodiment using a polyethylene disc. Generally, pore size may range from 40-50 microns. However, a plastic sheet having holes of approximately .030 inch in diameter may also be used. In a preferred embodiment, the porous member is located above and in physical proximity (which may include physical contact) to the reagent and serves as a means of both physically separating the reagent from the aqueous liquid which will be brought into contact with the upper surface of the porous member and of controlling the migration of gases from the area below the lower surface of the porous means to the upper surface of the porous member. In some embodiments, the reagent may be embedded in the porous means and the porous means may be immersed in aqueous solution to liberate the effervescent bubbles. The size of the pores is selected so as to initiate contact between the aqueous liquid and the reagent by inducing flow either via capillary action or by forcing liquid through the pores via hydrostatic pressure. The pore size also dictates the size of the effervescent bubbles which will be liberated. The hydrophilicity of the surface is selected so as to control the rate of flow of the aqueous liquid to the effervescent reagent and, thus, modulate the evolution of gas which is released through said porous means. For some applications, a buf¬ fer can be integrated into the porous member to control the final pH. For example, sodium bicarbonate and citric acid liberate gas in an acidic media. One may embed a basic solution such as tribasic sodium in the pores of the porous means to raise the pH of the medium in which the bubbles evolve prior to dispersion to the bulk aqueous solution. For other desired purposes other reagents may be included in the pores of the porous means.

The third component of the devices according to this invention is a reaction well which is of sufficient volume to contain the reagent and the porous member, as well as additional liquid and powder reagents (sample, buffers, etc.) The inside diameter of the reaction well and the outer diameter of the porous means are positioned such that a water-tight seal between the wall of the reaction well and the perimeter of the porous means is accom¬ plished. This may be achieved through an interference fit or bonding. As one skilled in the art will recognize, the amount of reagent needed to sufficiently mix the solution is dependent on the surface area of the reagent well and the amount of reagent used.

The method of the present invention comprises adding an aqueous solution to the upper surface of the porous means element and allowing the liquid to initiate contact with the effervescent powder of the reagent via either capillary action or hydrostatic pressure. Due to the watertight seal between the reaction well wall and the porous means, the only path through which the liquid may travel is through the pores of the porous means. As the liquid permeates the porous means, the water in the liquid comes in contact with the effervescent reagent and the chemical reaction is initiated resulting in the formation of effervescent bubbles. A dynamic state is then achieved, in which there is a movement of liquid through the porous means to the reagent, the water in the liquid interacts with the effervescent reagent, effervescent bubbles are produced and small bubbles are released through the porous means.

The timing of the initial bubble formation, size of the bubbles, and rate of bubble production all can be readily adjusted by various changes in the porous means including pore size, thickness, pore density, various chemical additives to the porous means and hydrophilicity of the surface. The desired result of these adjustments is to achieve desired modulation of the evolution of gases by the selection of the reagents and materials such as the porous means.

As noted above, the device comprises a powdered rea¬ gent which will liberate gas without the application of heat. Those skilled in the art will appreciate that there are many such compounds in use today, by way of example but without limitation, a preferred embodiment of the present invention is a reagent which liberate carbon dioxide when in contact with water, such as a blended powder consisting of citric acid and sodium bicarbonate.

As previously described, devices according to this invention contain a solid, porous means having pores gen¬ erally transverse to its upper and lower surfaces. The selection of the material to be used is important in controlling the timing of the initial bubble formation, the size of the bubbles, and the rate of bubble produc- tion. Those skilled in the art will appreciate the large selection of materials available for use, including with¬ out limitation artificial membranes, porous plastics, porous ceramics, and solid materials which have been modified to contain very small capillary holes. Those materials which are naturally hydrophobic can be modified to a predetermined or desired hydrophilicity by plasma treatment, corona discharge treatment, or treatment with a surfactant.

The following examples are presented to further illustrate particular embodiments of the present invention.

Example 1

A porous die cut high density polyethylene disc which is approximately 0.265" in diameter, 0.035" thick, and with a pore size range of 40-50 microns (Porex Technology) is placed in an 1.5M potassium phosphate tribasic aqueous solution containing 0.1% Triton X-100 and 5% ethanol. The solution and disc is first placed under reduced pressure for 5 minutes to evacuate all air which may have been trapped in the porous disc and to ensure that all disc surfaces are in contact with the solution and then an additional five minutes under atmospheric pressure. The treated discs are then dried by placing them in a vacuum oven at reduced pressure for 60 minutes at 70 degrees C. Approximately 15 mg of a citric acid and sodium bicarbo¬ nate blended powder (CIMA Labs) is then placed in the bottom of a reaction well 0.250" in diameter and 0.290" high and the dry, treated disc is press fit over the top of the powder; covering the powder in such a way so as to ensure that the only path which is readily available for liquid migration is through the disc pores. Two lyophil- ized reagent beads are then placed in the reaction well. The first bead consists of a lyophilized metal sol and the second bead consists of a lyophilized protein. A 140 microliter sample of human urine containing 25 ng/mL of phencyclidine is pipetted into the reaction well and is allowed to incubate for 5 minutes. The liberation of effervescent bubbles is delayed for approximately 10 seconds, thus allowing the lyophilized beads to partially reconstitute. After approximately 10 seconds, initial effervescent bubbles measuring approximately 0.010" in diameter are produced at rate of approximately 3 bubbles per second. The production of the bubbles continues for approximately 1.5 minutes, but at a steadily decreasing rate. At the end of the 5 minute incubation period, the liquid sample is transferred to a 1.2 micron, Biodyne & nylon membrane (Pall Biosupport) upon which has been immobilized antibody against phencyclidine in a discrete zone. After the liquid sample has been completely absorbed, the membrane is washed with an aqueous solution containing borate buffered saline and Lubrol at 0.02% w/v. The resultant colored discrete zone has a color intensity within the range of samples which are mechanically mixed.

Example 2

A die cut solid polystyrene disc which is approxi¬ mately 0.265" DA, 0.020" thick, and contains a series of four 0.030" in diameter holes located at 0.050" from center and spaced 90 degrees apart is placed in an aqueous solution of 0.01% Triton X-100 and 5% ethanol. The treated discs are then dried by placing them in a vacuum oven for 5 minutes at 70 degrees C. Approximately 15 mg of a citric acid and sodium bicarbonate blended powder (CIMA Labs) is then placed in the bottom of a reaction well 0.265" in diameter and 0.259" high and the dry, treated disc is press fit on to the top of the powder, covering the powder in such a way so as to ensure that the only path which is readily available for liquid migration is through the 0.030" diameter holes. Two lyophilized reagent beads are then placed in the reaction well. The first bead consists of a lyophilized metal sol and the second bead consists of a lyophilized protein. A 140 microliter sample of human urine containing 25 ng/mL of phencyclidine is pipetted into the reaction well and is allowed to incubate for 5 minutes. The liberation of effervescent bubbles is delayed for approximately 10 seconds, thus allowing the lyophilized beads to partially reconstitute. After approximately 10 seconds, initial effervescent bubbles measuring approximately 0.010" in diameter are produced at rate of approximately 3 bubbles per second. The production of the bubbles continues for approximately 1.5 minutes, but at a steadily decreasing rate. At the end of the 5 minute incubation period, the liquid sample is transferred to a 1.2 micron, Biodyne C nylon membrane (Pall Biosupport) upon which has been ^' immobilized antibody against phencyclidine in a discrete zone. After the liquid sample has been completely absorbed, the membrane is washed with an aqueous solution containing borate buffered saline and Lubrol at 0.02% w/v. The resultant colored discrete zone has a color intensity within the range of samples which are mechanically mixed.

Example 3

The porous polyethylene discs used in this invention were made hydrophilic by plasma treatment of the surface. Plasma treatment systems can be purchased by, for example, Plasma Science, Inc. , Belmont, California. The plasma treatment derivatizes the surface with functional groups which create a hydrophilic surface. Those skilled in the art will recognize that the plasma treatment of plastic is performed in a controlled atmosphere of a specific gas in a high frequency field. The gas ionizes, generating free radicals which react with the surface. For example, oxy¬ gen gas in a high frequency field results in positively and negatively charged monoatomic and diatomic oxygen ions, ozone, ionized ozone, oxygen atoms, metastably excited oxygen molecules and free electrons. The active species created are capable of reacting with the polymer carbon-carbon bonds to provide a variety of oxygen moie¬ ties on the surface. Analysis of the surfaces shows the presence of carbonyl, carboxyl, hydroxyl and hyperperoxide groups. The carboxyl and hydroxyl groups cause the sur- face of the polyethylene to become hydrophilic. The duration of the plasma treatment in the chamber of the device affects the degree of hydrophilicity of the sur¬ face. For example, treatment times ranging from 1 min. to 60 min. creates surfaces of varying hydrophilicity. Gen¬ erally, the power used is between 100 and 1,000 watts and the pressure is between 1 and 800 mtorr. The degree of hydrophilicity furthermore affects the degree of bubbling of the reaction mixture in the reaction cup; that is, the greater the hydrophilicity (the longer plasma treatment times, the greater the current and the greater the gas pressure) , the greater the bubbling ability of the discs.

Example 4

The porous polyethylene discs were made hydrophilic by adsorption of detergents to the surface. For example, the discs were placed in an ethanol solution containing 1% (w/v) Triton X-100 at room temperature for 30 min. The discs were removed and dried. The resulting discs were hydrophilic as evidenced by their ability to create bub- bles in a reaction mixture in the reaction cup. The degree of hydrophilicity of the discs was also changed by varying the Triton X-100 concentration in the ethanol solution from about 0.01% to 10% (w/v). Empirical adjustments may be made as desired for particular reagents, materials and purposes. The greater the Triton X-100 concentration, the greater the hydrophilicity of the discs as evidenced by their bubbling ability.

Example 5

Porous die cut high density polyethylene discs which are approximately 0.265 inches in diameter, 0.035 inches thick, and with a pore size range of 40-50 microns (Porex Technologies) are placed in a 0.1% or 10% Triton X-100 and 5% ethanol solution. The solution and discs are first placed under reduced pressure for 5 minutes to evacuate all air which may have been trapped in the porous disc and to ensure that all disc surfaces are in contact with the solution. Thereafter, they are placed for an additional five minutes under atmospheric pressure. The treated discs are then dried by placing them in a vacuum oven for 60 minutes at 70 degrees Centigrade. Approximately 15 mg of citric acid and sodium bicarbonate blended powder (CIMA Labs) is then placed in the bottom of a reaction well 0.250 inches in diameter and 0.290 inches high and deep. The treated disc is press fitted over the top of the powder in such a way so as to ensure that the only path which is readily available for liquid migration is through the disc pore. A series of assemblies are then completed with each of the two treated discs types and then the assemblies are labelled. 1.40 microliters of deionized^* water is added to each of the reaction well assemblies and the observed results are noted below:

1) the assemblies which were treated with the 0.1% Triton solutions began to bubble approximately 5 seconds after the deionized water was added. The bubbles were large, uniformly liberated one at a time, and no foam was generated;

2) the assemblies which were treated with the 10% Triton solution began to bubble immediately after the deionized water was added. The bubbles were very small and formed a layer of foam.

This best mode example demonstrates that control of the timing of the initial bubble formation, size of the bubbles, and rate of bubble production can be achieved by selecting the hydrophilicity of the surface by appropriate treatment.

While the invention has been described above in terms of specific embodiments, these have been provided for illustrative purposes only and are not to limit the scope of the invention which is defined by the claims.

Claims

1. Device for mixing an aqueous solution, comprising: a. reagent capable of liberating gas in the form of effervescent bubbles without the application of heat; b. porous means comprising pores generally trans¬ verse so as to permit liquid or gas to transverse there¬ through when said aqueous solution is added to said porous means; c. reaction well of sufficient volume to contain said aqueous solution, said reagent and said porous means, positioned such that upon addition of said aqueous solu¬ tion, said aqueous solution permeates said means, contacts said reagent, resulting in the evolution of gas which is released through said porous means to mix said aqueous solution.

2. Device of claim 1 wherein aqueous solution transverses through said porous means using capillary action.

3. Device of claim 1 wherein aqueous solution transverses through said porous means using hydrostatic pressure.

4. Device of claim 1 wherein said pores of said porous means are between 1 micron to 0.030 inches in diameter.

5. Device of claim 1 wherein said porous means is a microporous means.

6. Device of claim 1 wherein said reagent comprises citric acid and sodium bicarbonate.

7. Device of claim 1 wherein said porous means is high density polyethylene.

8. Device of claim 1 wherein said porous means is a ceramic disc.

9. Device of claim 1 wherein said porous means is positioned above said reagent.

10. Process for self-contained, time-released mixing employing a device comprising: a. reagent capable of liberating gas in the form of effervescent bubbles without the application of heat; b. porous means comprising pores generally trans- verse so as to permit liquid and gas to transverse there¬ through when said aqueous solution is added to said porous means; c. reaction well of sufficient volume to contain said aqueous solution, said reagent and said porous means; the process comprising adding said aqueous solution to said porous means in such a manner as to control deliv¬ ery of aqueous liquid to said reagent, and mixing said aqueous solution by the evolution of gas which is released through said porous ins.

11. Process of claim 10 wherein said pores of said porous means are between 1 micron to 0.030 inches in diameter.

12. Device of claim 10 wherein aqueous solution transverses through said porous means using capillary action.

13. Device of claim 10 wherein aqueous solution transverses through said porous means using hydrostatic pressure.

14. Process of claim 10 wherein said porous means is a microporous means.

15. Process of claim 10 wherein said reagent com¬ prises citric acid and sodium bicarbonate.

16. Process of claim 10 wherein said porous means is high density polyethylene.

17. Process of claim 10 wherein said porous means is a ceramic disc.

18. Process of claim 10 wherein said porous means is positioned above said reagent.

19. Device for mixing a solid in an aqueous solu- tion, comprising: a. reagent capable of liberating gas in the form of effervescent bubbles without the application of heat; b. porous means comprising pores generally trans¬ verse so as to permit liquid or gas to transverse there- through when said aqueous solution is added to said porous means; c. reaction well of sufficient volume to contain said aqueous solution, said reagent and said porous means, positioned such that upon addition of said aqueous solu- tion, said aqueous solution permeates said porous means, contacts said reagent, resulting in the evolution of gas which is released through said porous means to mix said solid through said aqueous solution.

20. Device for mixing an aqueous solution, comprising: a. reagent capable of liberating gas in the form of effervescent bubbles without the application of heat; b. porous means of predetermined surface hydrophil¬ icity comprising pores generally transverse so as to per- mit liquid or gas to transverse therethrough when said aqueous solution is added to said porous means; c. reaction well of sufficient volume to contain said aqueous solution, said reagent and said porous means, positioned such that upon addition of said aqueous solu¬ tion, said aqueous solution permeates said porous means, contacts said reagent, resulting in the modulated evolu¬ tion of gas which is released through said porous means to mix said aqueous solution.

21. Process for self-contained, time-released mixing comprising: a. providing a reagent capable of liberating gas in the form of effervescent bubbles without the application of heat; b. providing a device comprising a porous means of predetermined surface hydrophilicity comprising pores generally transverse so as to permit liquid and gas to transverse therethrough when said aqueous solution is added to said porous means and a reaction well of suffi¬ cient volume to contain said aqueous solution, said rea¬ gent and said porous means; c. adding said aqueous solution to said porous means in such a manner as to control delivery of aqueous liquid to said reagent, and mixing said aqueous solution by the modulated evolution of gas which is released through said porous means.

22. Device for mixing a solid in an aqueous solu¬ tion, comprising: a. reagent capable of liberating gas in the form of effervescent bubbles without the application of heat; b. porous means of predetermined surface hydrophil- icity comprising pores generally transverse so as to per¬ mit liquid or gas to transverse therethrough when said aqueous solution is added to said porous means; c. reaction well of sufficient volume to contain said aqueous solution, said reagent and said porous means, positioned such that upon addition of said aqueous solution, said aqueous solution permeates said porous means, contacts said reagent, resulting in the modulated evolution of gas which is released through said porous means to mix said solid through said aqueous solution.

23. Device of claim 1 or 22 wherein surface hydro¬ philicity is achieved by surface treatment.

24. Device of claim 23 wherein surface treatment is achieved by plasma treatment, corona discharge treatment or treatment with a surfactant.

25. Device of claim 1 or 22 wherein said porous means is naturally hydrophilic.