Device for precise chemical delivery and solution preparation Number:7,521,020 from the United States Patent and Trademark Office (PTO) owispatent A burette (10, 110, 200) suitable for delivery of a reagent into a target solution (50) employs diffusion for delivering the reagent. The reagent is in the form of a solution, which is combined with a matrix material (22), such as a gel or porous ceramic. A membrane (32) covers a delivery outlet (20) to the burette. In one embodiment, the delivery outlet comprises a plurality of fine bores (36), each one filled with or covered by a membrane (38). Stirring of the burette or target solution is achieved with a stirring means (104, 106). A heating or cooling means (80) heats a tip (16) of the burette.<H preparation, solution, Device, for, prec, 7521020.html, Patent, pto, 7521020

Title: Device for precise chemical delivery and solution preparation

Abstract: A burette (10, 110, 200) suitable for delivery of a reagent into a target solution (50) employs diffusion for delivering the reagent. The reagent is in the form of a solution, which is combined with a matrix material (22), such as a gel or porous ceramic. A membrane (32) covers a delivery outlet (20) to the burette. In one embodiment, the delivery outlet comprises a plurality of fine bores (36), each one filled with or covered by a membrane (38). Stirring of the burette or target solution is achieved with a stirring means (104, 106). A heating or cooling means (80) heats a tip (16) of the burette.

Patent Number: 7,521,020 Issued on 04/21/2009 to Gratzl, et al.

Inventors: Gratzl; Miklos (Mayfield Heights, OH), Tohda; Koji (Mayfield Heights, OH), Rozakis; George (Lakewood, OH)
Assignee: Case Western Reserve University (Cleveland, OH)

Appl. No.: 10/682,168

Filed: October 9, 2003

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09980089
PCT/US00/14805	May., 2000
60460082	Apr., 2003
60417149	Oct., 2002
60137134	May., 1999

Current U.S. Class: 422/100 ; 422/99; 436/180; 73/863

Current International Class: B01L 3/02 (20060101)

Field of Search: 422/99-101 73/863 436/180 424/468

References Cited [Referenced By]

U.S. Patent Documents


3993072	November 1976	Zaffaroni
4837161	June 1989	Stevens et al.
5008112	April 1991	DePrince et al.
6048457	April 2000	Kopaciewicz et al.
6050150	April 2000	Underhill et al.
6599754	July 2003	Miller et al.

Primary Examiner: Warden; Jill
Assistant Examiner: Nagpaul; Jyoti
Attorney, Agent or Firm: Tarolli, Sundheim, Covell & Tummino LLP

Parent Case Text

This application claims the benefit of U.S. Provisional Application Ser. No. 60/417,149, filed Oct. 9, 2002, U.S. Provisional Application 60/460,082, filed on Apr. 3, 2003, and is a continuation-in-part of U.S. patent application Ser. No. 09/980,089, filed on Jun. 26, 2003 now abandoned, and claims the benefit of PCT Application Serial No. PCT/US00/14805, filed May 30, 2000, and U.S. Provisional Application Ser. No. 60/137,134, filed May 28, 1999, from which U.S. patent application Ser. No. 09/980,089 claims priority, the specifications of all of which are incorporated herein in their entireties, by reference.

Claims

Having thus described the preferred embodiments, the invention is now claimed to be:

1. A delivery device for delivering a reagent into a target material comprising: a body which defines an interior chamber and a delivery port fluidly connected with the chamber, the chamber holding a reagent in solution; a membrane in fluid communication with the chamber and the delivery port, through which reagent passes when the delivery port is in fluid communication with the target material, such that substantially no volume change occurs in the target material during delivery of the reagent to the target material; and a matrix material dispersed throughout the body and being in contact with the membrane, the reagent diffusing through the matrix material to the delivery port; wherein the membrane includes a plurality of holes which define through passages through the membrane, each of the through passages being spaced apart from one another and separated by a rigid, impermeable portion of the membrane.

2. The delivery device of claim 1, wherein the body narrows intermediate the delivery port and the chamber.

3. The delivery device of claim 1, wherein the delivery port has a diameter of from about 10 to about 300 microns.

4. The delivery device of claim 1, wherein the membrane has a thickness of less than about 100 microns.

5. The delivery device of claim 4, wherein the reagent permeable membrane has a thickness of from about 2-50 microns.

6. The delivery device of claim 1, wherein the membrane is in or adjacent to the delivery port.

7. The delivery device of claim 1, wherein the holes have a diameter of less than about 300 microns.

8. The delivery device of claim 1, wherein the holes have a diameter of at least 100 nm.

9. The delivery device of claim 1, wherein the holes have a diameter of about 10-20 microns.

10. The delivery device of claim 1, wherein the holes are filled with a reagent permeable material.

11. The delivery device of claim 1, further including an agitator which agitates at least a portion of the body.

12. The delivery device of claim 1, further including a heating or cooling device which selectively heats or cools at least a portion of the body.

13. The delivery device of claim 1, further including a control system which calculates a delivery time for preparing a target solution of a preselected reagent concentration and volume.

14. The delivery device of claim 1, wherein the target material comprises a liquid.

15. The delivery device of claim 1, further including a hydrophobic coating on an exterior surface of the body.

16. The delivery device of claim 1, wherein the reagent diffuses from the delivery device into the target liquid with substantially no volume change in the target liquid.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a device and method for forming solutions. In particular, it relates to a diffusional burette for controlled delivery of chemicals and biochemicals into a target material, such as a liquid, and will be described with particular reference thereto.

2. Discussion of the Art

Currently available methods for reagent and solution preparation involve the use of traditional tools, such as analytical balances and glassware, including beakers of different sizes, pipettes, and burettes.

Such tools are suitable for preparing reagents and solutions of relatively large volumes, in the milliliter range. However, a demand has arisen for delivery devices which are capable of delivering liquid volumes which are up to several orders of magnitude smaller, to reduce waste and associated costs, and also to accommodate the ever increasing sensitivity of the instrumentation techniques that use these reagents and solutions. To satisfy the emerging needs for solution preparation in the microliter (.mu.L) volume range, pipettes based on liquid displacement by a precisely controlled volume of air have been introduced. For example, Eppendorf-type pipettes which use air/gas pressure and air/gas displacement for volumetric reagent transfer and delivery are now in use.

These devices deliver fixed or adjustable volumes of aqueous reagents from disposable plastic pipette tips by aspirating an appropriate volume of source solution into the tip and then delivering it into a target solution by reversing the air flow. A piston-type arrangement, inside the pipette body, whose air volume is precisely controlled, is used to meter the liquid volume that is aspirated into the tip, and subsequently delivered in one shot into the target solution.

For continuous (as opposed to bolus type) reagent delivery, mechanized piston burettes have been introduced, where a stepping motor controls the reagent volume aspirated, as well as delivered. Such mechanized burettes cover the microliter (mL) as well as .mu.L ranges in terms of delivered volume. For delivering sub-.mu.L volumes, different mechanized schemes have been conceived, such as a vibrating cantilever that "shoots" nanoliter (nL) droplets into the target across air.

While such novel pipette and burette designs reach into the .mu.L volume ranges, the mechanical working principles do not facilitate their adaptation to the handling of even smaller target liquid volumes or smaller reagent increments delivered. One constraint for the pipettes is evaporation, which becomes significant, even during short periods of time, for droplets smaller than about 1 .mu.L. Adjusting sub-.mu.L air volumes accurately and precisely is also difficult. Moreover, dislodging a nL-size droplet from a pipette tip is often difficult, since capillary forces become stronger relative to droplet mass as the droplet size decreases. Thus, accuracy and precision for the delivery of sub-.mu.L volumes by a pipette based on air displacement is not readily achieved.

Furthermore, preparation of solutions of relatively low concentrations often involves multiple steps that are difficult to perform with high final accuracy and precision. The first step is, typically, weighing a very small mass of solid (crystalline or powdered) material, i.e., the chemical that is to be present in the final solution, on an analytical balance. This, often tiny, amount of material may be hygroscopic (i.e., it absorbs water from humidity in air). This tends to falsify the weighed amount. Additionally, a powdery material may tend to float in air, contaminating the balance and the environment. The chemical may also be hazardous, posing problems to the user. The weighed material is then transferred into a beaker. These steps, requiring utmost care, are often sources of significant errors that propagate through all subsequent steps. Typically, multiple dilutions follow, until the desired low concentration is achieved. Further errors tend to be added in each step. In addition, such a procedure is labor intensive and prone to mistakes. Moreover, the process of serial dilution frequently uses far more of the raw material than is needed in the final solution than is needed, resulting in wastage of often expensive materials or hazardous waste production.

The mechanized burettes suffer similar drawbacks when sub-.mu.L volumes are to be delivered in a continuous fashion. Accuracy and precision of the displaced reagent volume become worse as the delivered volume decreases, especially in the nL volume range. Parasitic diffusion between the burette tip and the target liquid may add errors that are difficult to estimate, and even more difficult to correct. On the other hand, if the burette tip is not in direct contact with the target liquid, droplets form at the tip that must be dislodged to reach the target. Thus, delivery by the burette becomes effectively discrete.

The present invention provides a new and improved reagent delivery device and method of use and fabrication which overcome the above-referenced problems and others.

SUMMARY OF THE INVENTION

In accordance with one aspect of the present invention, a delivery device for delivering a reagent into a target material is provided. The delivery device includes a body which defines an interior chamber and a delivery port fluidly connected with the chamber. The chamber holds a reagent in solution. A reagent permeable membrane is provided through which reagent passes into the target material when the delivery port is in fluid communication with the target material. Substantially no volume change occurs in the target material during delivery of the reagent to the target material.

In accordance with another aspect of the present invention, a method of forming a target solution is provided. The method includes loading a reagent solution into a chamber fluidly connected with a delivery port. The delivery port is fluidly contacted with a target liquid for a period of time such that the reagent diffuses from the chamber through a porous material and delivery port into the target liquid to form the target solution.

In accordance with another aspect of the present invention, a diffusional burette for delivery of a reagent into a target material is provided. The burette includes a body which defines an interior chamber for receiving the reagent and a delivery port fluidly connected with the chamber, the delivery port defining a plurality of holes, each of the holes having a cross sectional width of less than 500 microns. A reagent permeable matrix material is disposed within the body. A solution of the reagent is in the chamber, such that the reagent diffuses from the chamber via the holes into the target material when the delivery port is in fluid communication with the target material.

As used herein, "delivery time" means the period of time in which a diffusional burette is in fluid contact with a target material such that a reagent passes from the diffusional burette to the target material.

An advantage of at least one embodiment of the present invention is that non-volumetric (non-convective) reagent delivery is used for transfer of chemicals. As a result, substantially no volume changes are induced in the target solution.

Another advantage of at least one embodiment of the present invention is that it enables automatic delivery to be achieved using natural processes that are spontaneously occurring during delivery.

Another advantage of at least one embodiment of the present invention is that the delivered amount may be controlled by monitoring the delivery time. Delivery time can be controlled extremely precisely using readily available techniques.

Another advantage of at least one embodiment of the present invention is that ranges of delivery rates are adjustable by varying the concentration of a chemical in a burette and by varying the geometrical dimensions of the burette.

Another advantage of at least one embodiment of the present invention is that high reproducibility and precision are achieved easily and cost effectively. As a consequence, waste disposal costs and reagent costs are minimized.

Another advantage of at least one embodiment of the present invention is that very low target solution volumes and delivery rates are feasible, enabling preparation of reagents of extremely low concentrations and/or volumes.

Another advantage of at least one embodiment of the present invention is that preparation of a reagent or solution can be achieved in one single step, without the need for serial dilutions.

Another advantage of at least one embodiment of the present invention is that a target solution can be prepared with multiple reagents by introducing the reagents simultaneously or sequentially.

Another advantage of at least one embodiment of the present invention is that it enables high precision and accuracy of the resulting reagent or solution to be achieved.

Another advantage of at least one embodiment of the present invention is that natural processes of diffusion, capillary forces, and surface tension, which affect delivery rates, are extremely reproducible, where other conditions are kept the same or accounted for, such as geometry, pressure, and temperature.

Another advantage of at least one embodiment of the present invention is that operator errors are minimized.

Another advantage of at least one embodiment of the present invention is that co-delivery of several chemicals is possible in a single step, thus leading to one step production of complex mixtures.

Another advantage of at least one embodiment of the present invention is that parallel processing of several or many deliveries into several or many solutions is feasible with little human control.

Another advantage of at least one embodiment of the present invention is that the delivery methods are amenable to microfabrication and MEMS technologies for serial production.

Another advantage of at least one embodiment of the present invention is that both aqueous and non-aqueous solutions can be prepared, using both aqueous and non-aqueous reagent sources.

Another advantage of at least one embodiment of the present invention is that the provision of a suitable membrane and/or matrix material inhibits convection or flow through a delivery port, such that a reagents is transported through the port primarily by diffusion.

Another advantage of at least one embodiment of the present invention is that it enables delivery to be achieved without moving mechanical parts.

Still further advantages of the present invention will be readily apparent to those skilled in the art, upon a reading of the following disclosure and a review of the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic side sectional view of one embodiment of a delivery device in a storage holder according to the present invention;

FIG. 2 is an enlarged side sectional view of the tip of the delivery device of FIG. 1;

FIG. 3 is a schematic side sectional view of a second embodiment of a delivery device according to the present invention;

FIG. 4 is an enlarged side sectional view of the tip of a burette according to a third embodiment of a delivery device according to the present invention;

FIG. 4A is an enlarged side sectional view of an alternative embodiment of a tip of a burette according to a fourth embodiment of a delivery device according to the present invention;

FIG. 5 is a side view of a delivery system including the delivery device of FIG. 1 with a dispensing device attached for delivery of a reagent into a target solution with a vibrator for mixing the target solution;

FIG. 6 is an enlarged view of an alternative embodiment of a dispensing device adjacent a cap of a burette having a barcode thereon;

FIG. 7 is a side view of an alternative embodiment of a delivery system including a stand for lowering a burette, an agitation means for vibrating the burette tip, and a support for a container of a target solution;

FIG. 8 is a perspective view of a fifth alternative embodiment of a delivery device according to the present invention, supported on a stand with a tip of the delivery device positioned in a target liquid;

FIG. 9 is a perspective view of the delivery device of FIG. 8, supported on a stand, after removing the tip from the formed target solution;

FIG. 10 is an enlarged sectional view of the tip of the delivery device of FIG. 8;

FIG. 11 is an enlarged top view of the membrane of the delivery device of FIG. 8;

FIG. 12 is a side sectional view of a sixth alternative embodiment of a delivery device according to the present invention, with a tip of the delivery device undergoing rotation;

FIG. 12A is an enlarged side sectional view of the tip of the embodiment of FIG. 12;

FIG. 12B is an enlarged bottom view of the membrane of the embodiment of FIG. 12;

FIG. 13 is an enlarged sectional view of the membrane of the delivery device of FIG. 12, illustrating the diffusion layer in a target liquid around a hole in the membrane, both with and without stirring, showing that the diffusion layer is reduced in thickness when stirring is used;

FIG. 14 is a perspective view of a pen-type delivery device according to a seventh embodiment of the present invention;

FIG. 15 is a perspective view of the pen-type delivery device of FIG. 14, being manually inserted into a target liquid;

FIG. 16 is a schematic view of a processor controlled delivery system according to another embodiment of the present invention;

FIG. 17 is a side sectional view of an eighth alternative embodiment of a delivery device according to the present invention;

FIG. 18 illustrates one method of preparing the delivery device of FIG. 17;

FIG. 19 is a side sectional view of a ninth alternative embodiment of a delivery device;

FIG. 20 is a side view of a delivery and monitoring system for evaluating the delivery devices according to the present invention;

FIG. 21 is a plot of cyclic voltammograms showing current in amps vs. electrode potential in volts for ferricyanide delivery times between 0 and 41.3 minutes;

FIGS. 22a, 22b, and 22c show plots of concentration of ferricyanide in a fresh target solution in mM vs. reagent delivery time in minutes for three sequential delivery procedures from the same burette;

FIG. 23 is a plot of potassium ion concentration in mM in a target solution measured by a K.sup.+ ion selective sensor delivered by a burette during continuous delivery;

FIG. 24 shows the dimensions of an exemplary burette;

FIG. 25 is a plot of potassium ion concentration in a target solution over time; and

FIG. 26 is a schematic view of a delivery port of a diffusional burette according to the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

With reference to FIG. 1, a delivery device in the form of a diffusional burette 10 suitable for the delivery of a chemical, chemical species, biological, or biochemical species or molecule (hereinafter referred to generally as a "reagent") to a target material or vessel is shown.

The target material is one which, on fluid contact with the burette 10, allows the reagent to be taken up or absorbed by the target material. Suitable target materials include liquids, such as water, aqueous solutions, and organic solvents, porous solid materials, such as medicament carriers, ceramics, and the like, absorbent fibers, semisolid materials, such as gels, and combinations thereof. While the target material will generally be referred to as being a liquid, it will be appreciated that other target materials are also contemplated.

Exemplary reagents include laboratory reagents, such as acids; bases; ions, e.g., alkali metal and alkaline earth metal ions, halide ions, and the like, such as potassium ions and bromide ions; reducing and oxidizing agents and agents that can be reduced or oxidized, such as ascorbic acid and permanganate ions, ferricyanide, ferrocyanide; drugs; pharmaceuticals; antigens; antibodies; protein molecules; and the like. The reagent is preferably in the form of a solution 11, which can be aqueous or nor aqueous. When the reagent diffuses into the target solution, it may form a target solution containing the reagent. Or, the reagent may undergo a reaction to produce a second species. Thus, the target solution need not contain the reagent or may contain less reagent than the amount actually diffused.

While the reagent is generally described in terms of a single chemical or species, it will be appreciated that a single burette 10 may deliver two or more reagents at the same time. These reagents may be in similar or different concentrations, according to the desired end use of the burette and either mixed together or separately contained within the burette.

The burette 10 of FIG. 1 includes a body or casing 12, formed from glass, plastic, ceramic, or other suitable material which is preferably impermeable to and unreactive toward the reagent and its solution. The body is preferably impermeable to and unreactive toward the target material. The body 12 includes a reagent holding portion 13, which defines an interior chamber 14 that holds a quantity of a reagent in solution, introduced via an opening 15 at one end of the chamber 14. The body also includes a tip portion 16, in fluid communication with the other end of the chamber 14, through which the reagent is delivered from the burette. The shape of the tip portion 16 affects the delivery, as will be described in greater detail below. A cap 17 or other sealing member closes off the opening 15 to the reagent holding portion 14 after filling with the reagent. Intermediate the tip portion 16 and the reagent holding portion 13 is optionally a shoulder portion 18. The shoulder portion may be conical and have an internal diameter D at its widest end 19 which is wider than the diameter d of a delivery port 20 in the tip 16. As shown in FIG. 1, the reagent holding portion 13, shoulder 18, and tip 16 are integrally formed, with the shoulder being simply an extension of the tip, although other configurations are also contemplated. FIG. 1 provides exemplary dimensions for the burette 10, although it will be appreciated that burettes of different sizes are readily formed. Preferably, the tip 16 has a substantially smaller interior diameter than that of the reagent holding portion.

In the embodiment of FIGS. 1 and 2, the tip 16 has the general shape of a bullet, which is conical with a delivery port 20 in the form of a small hole or holes at the end 21 of the tip, or near the tip end. The tip can be thus be a tapered portion of the body, which narrows between the reagent holding portion 13 and the delivery port 20. Alternatively, the tip has a cylindrical shape or other suitable shape. The size of the port 20 can be from about 10 microns to about 10 mm in cross sectional diameter, depending, in part, on the desired delivery rate, although larger or smaller dimensions are also contemplated. In one embodiment, the port 20 is from 10 to about 500 microns in interior diameter.

In the embodiment of FIG. 2, the delivery port 20 extends perpendicular to the longitudinal axis x of the body. It is also contemplated that the delivery port may be angled to the longitudinal axis x of the body, as shown in FIG. 4. This helps to reduce any tendency for air bubbles to become trapped in or around the delivery port.

In the embodiment of FIG. 1, the reagent holding portion 13 holds a filling comprising the reagent, optionally a solvent in which the reagent is dispersed to form a reagent solution 11, and optionally a matrix material 22, through which the reagent and solvent are able to permeate. This ensures that there is substantially no convection inside the burette. The matrix material is a solid or "semi-solid", such as a gel or a porous material (e.g., porous metals, such as inert metals e.g., nickel, titanium, and stainless steel, inert porous non-metallic materials, such as ceramic and glass, fibrous materials, such as fibrous glass, and combinations thereof) that contains the reagent solution 11. Suitable gels include agarose, polyacrylamide, other hydrogels, hydroxyethyl methacrylate (HEMA), and the like. The matrix material is preferably electrically neutral, and of low enough density to allow the reagent to permeate therethrough.

To avoid interactions with the reagent, the matrix material is preferably chemically inert towards the reagent. In one embodiment, the matrix material comprises a gel having a permeability which can be modulated via an electric voltage, voltage gradient, or other imposed electrical property. The delivery rate can thus be varied from external to the burette by a voltage controller or the like (not shown).

The gel helps to retain the reagent solution 11 within the burette until the tip of the burette makes contact with the target liquid. The gel allows the reagent to permeate therethrough.

The reagent is preferably in the form of a solution 11, such as an aqueous solution or non-aqueous solution. The reagent may be intimately mixed with and/or absorbed by the matrix material 22. Alternatively, or additionally, the matrix material is disposed between the reagent solution and the delivery port, as discussed in greater detail below. In an alternative embodiment, the matrix material is omitted.

The concentration of the reagent in the solution 11 depends, in part, on the desired concentration or concentration range of solutions to be prepared with the reagent. The concentration of the reagent can also be varied, dependent on the desired delivery time. Preferably, a plurality of burettes are provided, each one having a different concentration range, such that a user can select an appropriate burette according to the desired amount delivered, concentration, or concentration range. Alternatively, or additionally, other aspects of the burettes are modified to provide different absolute delivery rates and/or reagent, such as delivery port characteristics. For example, the target volume and desired concentration are first ascertained. From these values, the total amount to be delivered can be determined. The desired accuracy and precision of the delivered reagent and optionally the accuracy of tracking delivery time can also be taken into account in determining an optimum delivery rate. In some instances, such as in a titration or buffering in the target solution, the exact amount to be delivered is not known. In such instances, the amount delivered can be determined from the delivery time. Additionally, in some cases, the amount to be delivered cannot be determined from the volume of the target solution and the desired concentration. For example, where the reagent is undergoing a reaction as it is being delivered, such as in a titration, the desired delivered amount is dependent on the concentration of the corresponding reactant and/or the reaction end point. For example, the desired concentration of reactant in the target solution at the end point may be zero. Even though much larger amounts are actually delivered, the reactant is consumed. Similarly, where buffers are present, the amount delivered may be well in excess of what would be calculated from the final target concentration.

With reference now to FIGS. 2 and 4, a reagent permeable material 30 is optionally positioned in or adjacent the tip 16 of the burette, through which the reagent passes when leaving the burette. In this embodiment, the gel 22 may be omitted. As shown in FIG. 2, the permeable material 30 may be in the form of a porous membrane or layer 32, formed from cellulose, modified celluloses, such as acetyl cellulose or nitrocellulose, polyurethane, or the like. The membrane may be in the form of a single layer, as shown in FIG. 2, or multiple layers, as described in greater detail below. The membrane 32 may be about 2-100 microns in thickness, more preferably, less than 50 microns, most preferably, about 5-30 microns in thickness. FIG. 2 shows the membrane covering the tip end 21 and thus defining the delivery port 20, although it will be appreciated that the membrane may be positioned elsewhere in the tip 16 or shoulder 18 such that it provides a permeable member, which acts as a conduit, through which all of the delivered reagent diffuses from the burette.

In one embodiment, the membrane 32 is formed from a gel which has an electrically controllable property, such that the rate of diffusion can be controlled electrically, by an external device, as described above for the matrix material.

In yet another embodiment, the membrane 32 is formed from a material having holes in the form of pores 36, such as a thin layer of lipophilic or hydrophilic material, which may be 6-20 microns in thickness. The pores may be of micron or submicron diameter. For example, the pores may be from 0.01 to 100 microns in diameter. In one embodiment, the pores are at least about 0.1 microns in diameter. In one embodiment, the pores are less than about 10 microns in diameter. The pores 36 may be of uniform size or different sizes. An exemplary membrane material is a cyclopore membrane available from Whatman which has a clear and reproducible pore structure, although non-uniform pore sizes are also contemplated. Although having pores results in small semispherical volumes at the pore openings where concentrations are not uniform, the concentration at about 10-20 microns from the pores becomes substantially uniform due to the large number and small sizes of the pores. The pores thus produce a steady state delivery similar to a microelectrode array, with very little flow dependence on either side of the delivery port (flow in the target liquid or inside the burette body).

Specifically, the pores result in semispherical non-uniform volumes. This means that a local larger concentration exists at the center where the pore opening is located. A decaying concentration occurs along radial distance from the pore opening, so that local concentration depends on the distance from pore opening. If the semispherical concentration decays do not appreciably overlap, then the individual pores each deliver at steady state, which is independent of each other. If the pores are small enough, the pores produce small enough semispherical volumes that are too small for a modest flow to disturb, leading to constant delivery rate independent of small macro flows. The delivery rate is thus constant, after a negligible initial transient time during which the semispherical volumes form.

In another embodiment, shown in FIG. 4, the membrane 32 is from a rigid substrate 34 of a polymeric material, ceramic or the like, in the shape of a generally cylindrical body spanning the interior of the tip 16. The substrate 34 has a plurality of outlets 35, which together define the delivery port 20. Specifically, the substrate 34 is penetrated by a plurality of fine holes, such as bores 36 (FIG. 4) extending through the substrate between outlets 35 and inlets 37. The width of each of the bores 36 is dependent, to some extent, on the desired delivery rate and the method of forming the bores. For example, the bores 36 may in the general shape of hollow cylinders, although it is also contemplated that the bores may define convoluted paths or have different cross sectional shapes. The bore may be of about 0.1-300 microns in diameter. In one embodiment, the bores are about 10-200 microns in diameter. For example, the substrate 34 may contain 3, five, ten or more of such bores 36. In one embodiment there are from about 10-1000 bores. The bores 36 can be filled with a porous material 38 similar to that used for the matrix material 22 or membrane 32 in FIG. 2. As shown in FIG. 4, the membrane 32 is positioned at the tip end 21, although it is also to be appreciated that the substrate may be positioned elsewhere in the tip 16 and/or in the shoulder. It will be appreciated that the substrate 34 may be formed integrally with the tip 16 or be formed as a separate element, which is positioned in the tip.

In one embodiment, the bores 36 are all of the same width or of approximately the same width (e.g., at least about 90% of the bores are within .+-.20% of a median width of the bores, and can be within .+-.10% of a median width of the bores). In another embodiment, the bores are of different widths.

In the embodiment of FIG. 2, the bores are aligned with the longitudinal axis of the body. It is also contemplated that the bores 36 may be angled to the longitudinal axis, particularly where the delivery port is angled to the longitudinal axis, as illustrated in FIG. 4a.

It will be appreciated that similar delivery rates can be achieved by reducing the number of bores and at the same time, increasing their diameter. For example, 10-30 bores of about 20-70 micrometers in diameter may yield a similar diffusion rate to about 3-8 bores of 100-200 micrometers in diameter.

To achieve bores 36 of suitable fineness, an Excimer laser may be used. The laser allows a single hole 36 or plurality of holes to be formed in a substrate 34 with a high degree of reproducibility--i.e., the bores are virtually identical. Making techniques can be used to define the locations of the holes to be drilled. In one embodiment, the laser is used to "drill" holes in a plastic or other polymeric material, such as a polyimide, (silicone) rubber, or polytetrafluoroethylene (Teflon.TM.) material in the form of tubing or a sheet. The tube or sheet may be cut to the appropriate size for the substrate before or after drilling the holes. Excimer lasers are capable of forming bores of as little as about 10 microns in diameter, although bores of about 50 micrometers or greater are generally satisfactory.

Another method of forming holes 36 is to use silicon microfabrication technologies to produce the holes in a silicon or similar substrate 34. Such a substrate need not cover only the tip delivery port, it may be a large part or all of the burette body.

In one embodiment, the bores 36 are distributed in a ring, away from the center of the port. This allows for a more uniform and rapid dispersal of the reagent in the target solution, particularly when the tip is rotated, as is discussed below.

The burette 10 is intended to be disposable after use, although it is also contemplated that an empty burette may be refilled with reagent. In a preferred embodiment, large numbers of burettes are prepared by a supplier and shipped to facilities where the burettes are to be used. In one embodiment, each burette is used to prepare only a single target solution and is then disposed of.

The reagent is delivered from the burette into a target liquid by diffusion. This results in no or substantially no volume change in the target liquid during the addition of the reagent. By "substantially no volume change," it is meant that the target liquid does not increase or decrease in volume by more than 5% of the volume of the reagent solution 11 equivalent to the amount of reagent being transferred. More preferably, the volume change is no greater than 1% of the volume of the reagent solution equivalent to the amount of reagent being transferred. For example, if the reagent is in solution at a concentration of 1 Moles/L (1M) in the burette and it is desired to deliver 1 Moles of the reagent to the target liquid (i.e., the equivalent of 1 mL of reagent solution), the target liquid increases or decreases in volume by no more than about 0.05 mL, and preferably, by no more than 0.01 mL during the delivery time.

It will be appreciated that decreases in volume of the target solution can arise due to osmotic pressure. The high relative concentration inside burette causes water to flow into the burette due to the osmotic pressure difference. In general, the extent of water flow depends on the actual osmotic pressure difference between the target liquid 50 and the reagent solution 11 as well as on the characteristics of any membrane that separates the two. In practice, the effect of osmotic pressure upon target volume is negligibly small where the delivery time is in the minutes range, or less.

In one embodiment, the water (or other solvent) flowing between the burette and the target solution accounts for less than 10%, more preferably, less than 5%, and most preferably, less than about 1% of the equivalent volumetric delivery rate. The equivalent volumetric delivery rate F can be defined by the expression F=R/C.sub.R where R is the reagent delivery rate in moles per unit time and C.sub.R is the concentration of reagent inside the burette. F depends on the delivery port characteristics and the reagent concentration inside the burette.

For example, if the reagent delivery rate is 1 micromole/second and the reagent concentration in the burette is 1 M then the equivalent volumetric delivery rate would be 1 micromole/s per 1 mmole/mL=10.sup.-3 mL/s=1 microL/s. Where delivery is achieved within a few minutes of operation, the effects of osmosis are negligible. Preferably, no more than about 10%, more preferably, less than 5%, most preferably, less than about 1% of this value will be water flow, i.e., in the example above, a volume change of less than 0.1 .mu.L/second, more preferably, less than 0.05 .mu.L/second, most preferably, less than 0.01 .mu.L/second is due to water flow. In practice, the volume change due to water flow is typically in the direction from the target into the burette, and is typically far less than 5% of the equivalent volumetric delivery rate. In some cases, there is effectively no change in volume of the target solution, since the rate of reagent delivery from the burette to the target solution is approximately balanced by the water flow from the target solution to the burette.

To form the burette 10 of FIG. 1, the reagent solution 11 of choice is mixed with a gel forming material, such as agarose powder or other polymer forming material which is then loaded into to the chamber 14 and the gel allowed to set. The cap 17 is then screwed or otherwise attached to the open end 15 of the burette. The reagent remains within the burette, trapped by the gel until the tip 16 is placed in contact with the desired target solution.

Where a solid matrix material such as a porous ceramic is used, the porous material is immersed in or otherwise contacted with the liquid reagent solution 11. Vacuum infiltration techniques may alternatively be used to introduce the reagent solution to the porous material. The porous ceramic may be loaded into the chamber 14 prior to introduction of the reagent solution or loaded after introduction. Or, as described in greater detail below, the body 12 may be formed around the infiltrated porous material or gel/reagent solution mixture.

If no matrix material is used, the reagent solution 11 is introduced directly to the burette chamber. In this case, the membrane 32 is sufficiently resistant to the passage of the reagent solution therethrough that there is substantially no escape of the reagent from the body until it is able to move by diffusion into a target liquid in contact with the membrane. Where the matrix material and reagent solution are to be inserted separately, the matrix material can be added first and allowed to set before the reagent solution is introduced.

The burette 10 may be used immediately after formation or shipped and/or stored for a period of time prior to use. Accordingly, multiple burettes can be prepared and used as needed. The burette 10 holds the reagent solution until the tip is placed in fluid contact, generally in direct contact, with a target liquid 50 in which a target solution containing the reagent is to be formed (FIG. 5). Contact is maintained with the target liquid for a sufficient time (the delivery time) to form the target solution. This operation may be carried out manually, by an operator holding the burette and contacting the target liquid with the tip, or mechanically, as described in greater detail below.

In one embodiment, the burette 10 contains sufficient reagent to form a plurality of target solutions. It will be appreciated, however, that with each additional delivery, the concentration of the reagent in the burette tends to decrease, since the volume of solution in the burette remains essentially constant. Accordingly, the delivery time for second and subsequent deliveries is generally increased for forming corresponding target solutions of the same concentration and volume. The delivery time for a particular delivery in a sequence of such deliveries can be calculated, either empirically, or by using mathematical formulae, as will described in greater detail below.

Optionally, a dispensing device is provided for the burette 10. In one embodiment, shown in FIGS. 3 and 5, a dispensing device 40 selectively holds a burette in a sleeve 42 or other receptacle. A fresh burette 10 is inserted into the sleeve and a timing device, such as a clock 44 is reset to zero or to a desired delivery time, e.g., 10 seconds. The resetting of the clock may be carried out automatically by the burette pressing on a reset button 46 during insertion into the sleeve.

When the burette 10 is to be used to form a target solution, the tip 16 is placed in the target liquid 50 into which the reagent is to be delivered (FIG. 5) and the user simultaneously presses a button 52 on the dispensing device to start the clock 44. The reagent flows through the membrane into the target liquid. When the clock indicates that the desired delivery time has elapsed, the user removes the burette 10 from the solution, halting the reagent flow. Alternatively, or additionally, an alarm 54, such as a buzzer or flashing light, indicates when a desired delivery time has elapsed to alert the user to remove the burette tip 16 from the target solution. When the burette 10 is depleted, it is removed from the dispensing device 40 and the dispensing device can be reused with a fresh burette.

In one embodiment, shown in FIG. 6, the user uses a keypad 60 or other input device to instruct the timer device or a control system, such as a microprocessor controller 62 associated with the timer device 44 how much reagent is to be delivered. The control system is programmed with information concerning the delivery rate of the reagent from the burette 10 and other factors affecting delivery, such as the amount of reagent which has already been delivered from the device. The control system 62 determines how long it will take to deliver the desired amount of reagent and sets the clock 44 accordingly. In one embodiment, the control system interrogates the user by means of a screen 64 and the user inputs information which affects the delivery rate, e.g., via the keypad 60. For example, the control system 62 may ask the user for the concentration of the reagent in the burette being used, or for information regarding one or more of the ambient temperature, the desired delivery amount, the target concentration and the volume of the target solution. The control system uses this information in determining the appropriate delivery time. The control system may also ask the user for the desired final accuracy and precision. Based on the input values, the control system recommends a particular burette to be used (i.e., one that has low enough delivery rate such that the total delivery time is long enough to be controlled precisely and accurately.

To accommodate the demands of a variety of consumers, it is contemplated that a series of burettes of different dimensions, sizes, shapes, and material are provided to accommodate different reagents, concentrations, delivery rates, and the like. Identifying information, such as reagent type, concentration, volume, and the like, e.g., in the form of a computer recognizable indicia, such as a bar code 70, may be provided for each burette 10. The bar code is preferably affixed to the burette 10 or to packaging associated therewith. The bar code may also provide other information, such as the date of fabrication and filling, delivery rate, information for programming a timer device, and the like. Other recognition information may be provided, so that a user may keep track of the amount of reagent that had been delivered from a particular burette, allowing an adjustment of the calibration to be automatically made, if appropriate.

In one embodiment, shown in FIGS. 6, 7, and 16, the control system 62 is programmed to recognize the bar code 70 using a bar code reader 72. The control system optionally informs the user about the particular burette or makes calculations based on stored information about the burette which corresponds to the bar code. The bar code may be printed on an inside surface of the burette, to protect the bar code or some other condensed digital code system from outside dirt or contamination or obscuring effects or to ensure confidentiality of the information. The control system may also display, for example, which one of the different burettes of identical type should be used for delivering a given amount with a given precision. This is because the longer the delivery time (i.e., the slower the delivery rate), the higher the relative precision of the total amount delivered. The control system may include look-up tables, algorithms, or the like from which it calculates a desired delivery time, delivery rate, or the like, based on information scanned from the bar code 70 and/or or operator input information.

In place of a dispensing device 40, with its own clock 44, as shown in FIG. 3, a stopwatch or other timing device 44 is alternatively used by the user to monitor the delivery time.

Diffusion rates depend, to some degree, on ambient temperature. Accordingly, particularly when extremely precise quantities are to be delivered, controlling the temperature and/or correcting for temperature helps to ensure accuracy. Thus, in one embodiment, a controlled temperature environment (such as is generally provided in wet chemistry laboratories) is employed for deliveries from the burette 10. Both the burette 10 and the target liquid 50 in to which the delivery is to be made are allowed to equilibrate in the controlled temperature environment prior to delivery. Alternatively, for example, where a controlled environment is unavailable, the target liquid and/or burette are brought to a preselected temperature (e.g., by heating or cooling) prior to delivering reagent from the burette. In yet another alternative embodiment, corrections are made to the delivery time depending on the detected ambient temperature. The effect of small temperature variations on the delivery characteristics is estimated by using physicochemical theory, or from prior empirical measurements.

FIG. 7 illustrates a dispensing device 40', where similar components to dispensing system 40 are labeled with a prime (') and new elements are given new numbers. In the embodiment of FIG. 7, a heating element 80, such as a resistance heater, provides local heating of the tip 16 and optionally all or part of the reagent holding portion to heat the reagent solution therein. This allows thermostatic control of the delivered reagent (and thus, the temperature of the delivery diffusion process) and hence a more reproducible delivery rate, particularly where ambient temperatures are prone to fluctuation. The heating element 80 may also provide different heating levels, making the delivery rate tunable. This can be used to increase or decrease the delivery rate (increasing the temperature increasing the delivery rate, decreasing the temperature decreasing the delivery rate).

In one embodiment, the heating element heats the entire delivery port and membrane and optionally also an adjacent portion of the target liquid 50. This ensures that those elements whose temperature may affect delivery rate to some degree are at the same, reproducible temperature. For example, a partial casing or screening of a volume fraction of the target liquid closest to the delivery port is effected to ensure that the volume fraction whose temp may have an effect on the resulting effective delivery rate is effectively thermostatted. The remainder of the target liquid may then be at a slightly different temperature, without there being a significant influence on the delivery rate.

It has been found that an approximately 10.degree. C. increase in temperature may result in a doubling in the rate of a number of physicochemical processes, such as diffusion rate. A heating element 80 at or near the tip 16 of the burette 10 thus allows a constant temperature, which is higher than ambient, during the delivery by heating it above the ambient temperature. Additionally, heating enables the user to achieve different delivery rates using temperature modulation of the diffusion rate. It is also contemplated that a constant temperature may be achieved by cooling the tip. If a very small volume is to be cooled (or heated) then this can be effectively achieved using a heating or cooling element 80 employing conventional technologies (for example, for cooling, an electronic cooling system or heat pump can be used).

The delivery from the burette 10 into a target liquid 50 is continuous, with a rate which can generally be as low as practically desired. For intermittent additions, such as multiple standard additions, the burette tip 16 is removed from the target liquid and returned to the target liquid for each addition. For introducing the tip to a target liquid, a support means, such as a stand 90 is optionally used to lower the tip 16, either manually or by an automated operating system 92 into the solution, as illustrated in FIG. 7. The automated system includes a processor 62' analogous to processor 62. An automatic sensing device 94, electrically linked to the operating system 92, optionally senses the time when the tip first contacts the solution. The automated sensor 94 may use electrical impedance for detecting contact. The timing mechanism 44' starts counting the time from that moment. At the end of the preselected delivery period, the operating system 92 withdraws the tip from the target solution. In one embodiment, the stand 90 includes a mechanical lifting device 96, such as a piston operated actuator, e.g., operated by a motor (not shown) for removing and inserting the tip 16 into the target liquid 50 by lifting/lowering the burette 10. The mechanical lifting device 96 can be controlled by processor 62' to remove the tip 16 from the target liquid when a desired delivery time has elapsed.

As shown in the embodiment of FIGS. 5 and 7, the target liquid 50 is preferably placed in a suitable container 100. The container is optionally formed from or lined with a containment layer 101 such as a silicone layer, for creating surface tension forces which contain the target liquid on as small area of the containment layer as an approximately hemispherical bead of liquid. Where the reagent solution is hydrophilic, containment layer 101 is preferably formed from a hydrophobic material, and vice versa. The container 100 is optionally positioned on a support 102. Optionally, a means for stirring the target liquid 50 or reagent is provided. In the embodiment of FIG. 5, the support 102, and hence the container 100 and target liquid 50, is vibrated by an agitator in the form of a vibrator 104, although other suitable stirring means are also contemplated, such as fine gas jets or bubbling or integrated MEMS devices. The vibrator stirs the target liquid, helping to maintain the homogeneity of the target solution as the reagent is added.

In another embodiment, illustrated in FIG. 7, the reagent in the burette is stirred. In this embodiment, the means for stirring includes an agitator, such as a vibrator or rotator 106, which causes the tip 16 or other portion of the burette 10 to vibrate, rotate or otherwise move. The vibration/rotation or other movement of the tip 16 stirs the reagent solution in the burette 10, maintaining its homogeneity in the burette as reagent is dispensed. The agitator 106 also causes the tip 16 to stir the target liquid 50, when the tip is placed in contact therewith. It will be appreciated that more than one means for stirring 104, 106 may be employed, either during or subsequent to a delivery. The stirring means 104, 106 is preferably under the control of the operating system 92.

The rotation of the burette 10 (or other form of agitation) results in an adjustable enhancement of the delivery rate (i.e., allows for reductions in the delivery time) and tends to reduce errors which may arise from unwanted spontaneous convection.

With reference now to FIGS. 8-11, a burette 10 similar to that shown in FIGS. 1 and 2 is shown. For convenience, similar elements are given the same numbers and new elements are given new numbers. The burette includes a housing 107, formed from plastic, or other suitable material which surrounds the body 12 and has an opening 108 at a lower end, through which the delivery port 20 is exposed (FIG. 10). The body is formed from an inert material, such as polyacetal resin, Teflon.RTM., or the like and holds a quantity of a reagent solution 11. A reagent permeable material 30 in the form of a membrane 32 closes the delivery port 20 and is permeable to the reagent. In one embodiment, reagent permeable material also includes a layer 109 formed from a matrix material, such as a gel, e.g., an agar gel or other material allows the reagent to pass therethrough by diffusion, although it is also contemplated that the gel may be mixed with the reagent solution, as shown in FIGS. 1-4. The membrane layer 32 can be formed from a substrate, such as a polyimide or other plastic material or an inert metal sheet or other inert membrane material which is impermeable to the reagent, except where holes 36 are provided. The membrane may be, for example, about 20-30 microns in thickness. As shown in FIG. 11, the membrane 32 has a plurality of holes 36 (five in the illustrated embodiment), through which the reagent passes. The holes may be about 10-100 microns in diameter, e.g., about 50 microns in diameter, or other suitable widths, as noted above, and may be a distance h of about 0.01 mm to about 2 mm apart. The many holes (bores- or pores in the case of a cyclopore membrane) are ideally as close to each other as possible (to reduce overall size) without causing the resulting semispherical nonuniform concentration domains to overlap. The remainder of the polyimide film 32 is impermeable to the reagent, such that the reagent diffuses from the burette via the holes. The holes 36 may be formed by drilling bores, e.g., with a laser, as described above. Or the membrane layer 32 can be formed with pores.

In one embodiment, illustrated in FIG. 12, a burette 10 similar to that of FIGS. 8-11 includes a tip 16 which is rotatable. The burette includes a housing 107, formed from plastic, or other suitable material, which surrounds the body 12 and has an opening 108 at a lower end, through which the delivery port 20 is exposed. The housing holds the burette body 12 and a motor 300 powered by a power source, such as a battery 310, which in the illustrated embodiment is mounted within the housing, above the motor 300, although other locations are also contemplated. Alternatively, a remote source of power, such as mains supply, is used to power the motor and is connected thereto by suitable electrical wiring.

The motor 300 is connected by a drive shaft 312 to the cap 17 of the burette and thus imparts rotational movement to the body 12 of the burette. A switch 314, mounted to the housing, controls the motor. The switch may be a simple ON/OFF switch, or it may be a variable switch, which allows the speed of the motor to be varied. The motor may rotate the body at a speed of from about 50 to about 20,000 rpm. In one embodiment, the motor rotates the body at from about 1000 to about 5000 rpm.

The body 12 includes a reagent holding portion 13, which defines an interior chamber 14 that holds a quantity of a reagent in solution 11, which is introduced to the chamber via an opening 15 at one end of the chamber 14. For example, the chamber may hold about 5 mL of reagent solution 11. The body also includes a tip portion 16, in fluid communication with the other end of the chamber 14, through which the reagent is delivered from the burette. FIG. 12A shows the tip in greater detail. Intermediate the tip portion 16 and the reagent holding portion 13 is a tubular portion 18. The tubular portion has an internal diameter which is generally constant from the tip to the reagent holding portion, although conical or other shapes are also contemplated. The internal diameter d of the tip, which in this embodiment, also corresponds to the diameter of the tubular portion 18, can be as for other embodiments of the burette. In one embodiment, the diameter d is about 5 mm. A membrane 32 covers the delivery port. The membrane can be as previously described, e.g., of about 10-50 microns in thickness and have a plurality of bores drilled therethrough or otherwise formed therein, similar to those illustrated in FIG. 4, or be a naturally porous material. The bores/pores provide a plurality of through passages between the interior of the body and the exterior. As shown in FIG. 12B, the bores 36 are located in a ring, spaced from the axial center of the membrane 32 and preferably closer to the exterior of the membrane than to the center of the membrane. In this way, the reagent is carried away from the tip 16 by the fluid flow pattern in the target liquid 50, induced by the rotation of the tip in the target liquid. Closer to the axial center of the tip, the flow pattern tends to result in a lower flow rate.

The membrane 32 is carried by a membrane fitting 320, which is shaped to connect with the tube 18. Specifically, the membrane fitting is internally threaded at 322 and engages corresponding threads 324 on an exterior of the tube 18. In this way, the membrane 32 can be removed and replaced if desired. The membrane is optionally in direct contact with a gel layer 109 as described for the embodiment of FIG. 11.

As illustrated in FIG. 12, ball bearings 330 or other suitable guide members are located within the housing 107, close to the tip 16 of the burette for guiding the tube 18 during rotation of the burette.

As shown in FIG. 12, the housing 117 may