GB2453585A

GB2453585A - A probe and method for liquid sampling

Info

Publication number: GB2453585A
Application number: GB0719979A
Authority: GB
Inventors: Patrick Douglas Shaw Stewart
Original assignee: Shaw Stewart P D
Current assignee: Shaw Stewart P D
Priority date: 2007-10-14
Filing date: 2007-10-14
Publication date: 2009-04-15
Also published as: GB0719979D0

Abstract

A method of loading at least one sample into a conduit by way of a probe 40 where steps are taken to ensure that an immiscible fluid 12 remains between the probe and the sample. This may be achieved in any of three embodiments: i) wherein an immiscible liquid is provided covering the sample 10, moving the probe such that its orifice is below the level of the immiscible fluid and close to, but not touching, the surface of the sample liquid, and aspirating liquid sufficiently fast that both sample liquid and immiscible liquid are drawn into the bore of the probe; ii) covering each sample with an immiscible liquid, moving the probe so that its tip is below the surface of the sample, aspirating liquid into the probe before the probe makes contact with the sample liquid and raising the probe above the surface of the sample; iii) covering the sample with an immiscible liquid, providing a probe that possesses a main bore for aspirating the sample and at least one additional bore for ejecting the immiscible liquid, moving the tip of the probe to below the level of the sample and aspirating liquid. A probe in the form of a tube possessing a flared, chamfered or radiused tube is also disclosed. The method preferably helps prevent contamination in sampling liquids for the analysis, synthesis, and crystallization etc. of samples.

Description

New and improved probe and method for liquid sampling

TECHNICAL FIELD

There are many applications of liquid handling where it is necessary to pick up a plurality of samples so that each sample can be treated or analyzed. This invention relates to sampling systems for such applications which may be applied to clinical, biological, chemical, forensic, physical etc. applications.

The invention is suitable for microfluidic systems where samples are moved as droplets in an immiscible carrier liquid, and also for macro-scale liquid handling applications.

BACKGROUND OF THE INVENTION

Reichler eta!., in U.S. patent number 4121466, disclosed a probe for aspirating solutions where an immiscible liquid was placed upon the outer and inner surfaces of the aspirating probe of a dispensing system. These inventors used a coaxial liquid dispenser to apply the immiscible liquid, such as silicone oil, onto the outer surface of the probe. Some of the immiscible liquid is also drawn into the interior of the probe, where it coats the inside surface of the probe, preventing the probe from coming into contact with the samples that are aspirated. The exterior and interior surfaces of the probe are chosen to have a high affinity with the immiscible liquid, and a low affinity for the samples to be aspirated. A disadvantage of this system is that it is difficult to maintain the film of carrier liquid at the tip of the probe. Therefore contamination remains a problem.

In 1957 Leonard Skeggs invented a special flow analysis technique named "continuous flow analysis" (CFA) that was commercialized by Technicon Corporation. This method is disclosed by Skeggs in US patent number 2797149, issued in 1959. Originally CFA used air to segment the flow of reagents and samples, but the air was sometimes replaced by an immiscible carrier liquid as disclosed by Smythe and Morris in e.g. US3479141, issued in 1969. As long as the droplets and carrier fluid are moving, a film of carrier fluid can be maintained on the walls of the conduits. Thus the carrier liquid can prevent the sample and reagent droplets from touching the walls of the conduit system through which they pass, preventing contamination of the walls and the subsequent samples. US3479141 also teaches the injection of solution into droplets at a T-junction, which is a useful technique for modem microfluidic applications.

Figure 3 of US3479 141 indicates the injection of copper neocuproine solution into droplets to assay glucose in the droplets. Note that droplets are here treated as a continuous stream. Also, samples are distributed over several droplets. Therefore droplets are not individually treated or manipulated.

A similar approach was developed by the inventor of the present patent in GB patent 2097692B in 1982.

Here samples etc. are dispensed into oil in a conduit system as individual droplets. Liquids can be added to samples, which can be heated, incubated etc. Each droplet is equivalent to a test-tube and it can be dealt with individually. Also, GB2097692B teaches miniaturization and construction of conduits by forming depressions in the surface of one or more sheets and bringing these sheets into face-to-face contact.

More recently microfluidic approaches have become commercially important. Some of these approaches are similar to GB2097692B because they move droplets in immiscible liquids through conduits. For example, Ismagilov disclosed in W02004038363 (2006) the simultaneous introduction of a plurality of liquids, through one or more orifices, into an immiscible fluid in a conduit.

Such droplet-based microfluidic devices have potential applications in which it is essential to reduce contamination between droplets as much as possible. For example, potential applications include molecular diagnostics, where the polymerase chain reaction is used to "amplify" DNA. If a single DNA molecule were to be transferred from a droplet to a subsequent droplet, a false positive result could arise.

By using detergents and ensuring that the droplets and carrier liquid do not remain stationary in the conduit at any time, contamination of the main conduits can be avoided. However, the device that loads the samples into the conduit from e.g. a 96-well plate remains a problem, because contamination of the probe is likely to occur, as noted above.

The objective of the present invention is to provide a sampling device that can load droplets into a conduit filled with a carrier liquid without significant contamination of droplets by materials left behind by previous droplets.

BRIEF SUMMARY OF THE INVENTION

One aspect of the invention is the avoidance (or reduction) of contamination of sample droplets at the time of sample aspiration.

Another aspect is an approach to aspirating samples into a probe where a film of immiscible liquid is maintained between said probe and said samples.

The invention relies on a liquid that covers samples prior to picking up droplets of sample from receptacles. This liquid must be at least partially immiscible with the samples. This immiscible liquid will be referred to hereinafter as the "covering" liquid. The invention can advantageously be used with a microfluidic system that uses a carrier liquid to move droplets in a conduit system. The covering liquid can be either miscible or immiscible with the carrier liquid (but both must be at least partially immiscible with the samples).

A first alternative sample aspiration method comprises covering the sample with an immiscible liquid, moving a probe very close to the surface of a sample without touching it, then rapidly withdrawing liquid into the probe thereby sucking into the probe both covering liquid and a droplet, slug or plug of sample.

A second alternative sample aspiration method comprises moving a probe rapidly down through a covering liquid and into a sample so that a film of covering liquid is preserved around said probe, and then withdrawing liquid into the probe so that the liquid sample together with some covering liquid is sucked upwards into the bore of the probe, then rapidly withdrawing the probe.

A third alternative sample aspiration method comprises providing a probe with a plurality of holes around a central hole, and ejecting covering liquid through said plurality of holes while aspirating a sample and some covering liquid into said central hole.

For normal operation, the covering liquid must be less dense than the samples to be aspirated. (An inverted version of the invention that uses a denser "covering" liquid is also possible, where samples are withdrawn from below. In such a case some instructions and geometrical descriptions in this patent must be reversed because moving downwards would become upwards and vice versa.) The apparatus of the invention comprises a tube, needle or bore that can be moved into a receptacle containing a sample. The probe can advantageously possess a flange around its extremity and can possess flared, chamfered or radiused corners. Instead of a flange, a probe with an extra-thick wall can be used.

Another embodiment possesses at least one additional bore close to the main bore of the probe, where the additional bores are suitable for ejecting covering liquid.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures Ia to Id show a first sequence for loading a sample into the probe.

Figures 2a to 2d show an alternative second sequence for loading a sample into a probe.

Figures 3a to 3d show an alternative third sequence for loading a sample into a probe.

Figures 4a to 4g show six alternative designs for the probe.

Figures 5a and 5b show two probes that are equipped with metal conductors (60) that can be used for capacitive level-detection.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail, with reference to the accompanying drawings.

In the accompanying drawings, like features are denoted by like numerals.

All figures show schematic cross-sections through various devices.

Figures Ia to Id show a first sequence for loading a sample into the probe.

Figure Ia shows a sample (10) that is covered by an immiscible covering liquid (12) in a receptacle (14).

Above the receptacle is a probe (16).

Typically the sample will be aqueous and the covering liquid will be an oil, such as paraffin oil or silicone oil. Fluorinated oils that are denser than water cannot normally be used.

Figure lb shows that the probe (16) is lowered into the covering liquid (12) until it is close to the sample (10), but does not touch it.

Figure Ic shows that liquid is withdrawn into the probe sufficiently fast that a tongue of sample (30) is lifted up and flows towards the probe. (Covering liquid also flows into the bore of the probe.) Figure id shows that the probe rises and liquid is again withdrawn into the probe so that the sample passes into the bore (40) of the probe where it forms at least one droplet (42) that moves along said bore as shown.

The probe may now move to another sample, and the process can be repeated until all desired samples have been loaded.

Note that a film of the covering liquid is maintained between the sample and the probe so that the probe never comes into contact with the sample. Contamination of the probe and samples is thereby reduced or eliminated.

In figure 2a a probe is positioned above a sample that is again covered with a covering liquid. (The probe is shown with a flange, as described below with reference to figure 4c. However, such a flange is not essential, and indeed any of the probe designs described below could be used.) In figure 2b, the probe moves down until it is below the surface of the sample. Note that a film of immiscible liquid (20) remains between the probe and the sample.

In figure 2c, liquid, including a tongue of sample (30) and covering liquid is aspirated into the probe.

Figure 2d shows that the probe rises and liquid is now withdrawn into the probe so that the sample passes into the bore (40) of the probe where it forms at least one droplet (42) that moves along said bore as indicated by the arrow.

Figures 3a to 3d show an alternative third sequence for loading a sample into a probe. This method relies on a probe that has additional bores that open close to the bore that aspirates sample. These additional bores are used to eject covering liquid at or near the end of the probe. Such a probe is described below with reference to figures 4e and 4f, and also 4g.

In figure 3a the probe with additional bores (57) is positioned above a sample (10) that is again covered with a covering liquid (12).

In figure 3b. the probe moves down until its tip is below the surface of the sample. Covering liquid is ejected from the additional bores as indicated by the arrows. This ensures that the sample (10) does not come into contact with the probe.

In figure 3c, liquid, including sample and covering liquid is aspirated into the main bore of the probe (40) as indicated by the arrow pointing upwards. Advantageously, carrier liquid continues to be ejected from the additional bores while this occurs.

Figure 3d shows that the probe rises and liquid is withdrawn into the probe so that the sample passes into the main bore (40) of the probe where it forms at least one droplet (42) that moves along said bore as indicated by the arrow.

Figures 4a to 4g show six alternative designs for the probe.

Figure 4a shows a plain tubular probe (50).

In figure 4b, the probe possesses a chamfer or flare (52) at the entrance of the probe, similar to the probe of figures lato Id.

The next two alternatives possess an enlarged flat surface (54) around the bore of the probe, which can improve sampling. Figure 4c shows a probe with a flange (56). In figure 4d the probe comprises a thick-walled tube (58) with a flat surface at the end. These two designs discourage the covering liquid from flowing downwards into the probe and generally restrict the flow of covering liquid. This encourages sample to flow towards the main bore of the probe.

Figure 4c also shows that the main bore can be radiused (53) where it meets the outside surface, and figure 4d shows that the outside corner of the probe (55) can be chamfered or radiused.

Figure 4e shows an example of a probe that can be used with the method shown in figures 3a to 3d. It possesses additional bores (57) in addition to a central main bore (40). The central bore is used to aspirate sample, while the additional bores are used to eject covering liquid. Figure 4f shows a cross-section through the probe of figure 4e along the line A-B. (Figure 4f shows six additional bores (57), but other numbers of bores can be used. Advantageously there should be at least two bores, arranged symmetrically.) Figure 4g shows the end of an alternative probe that has a circular opening (59) for the ejection of covering fluid. Such a probe could for example be formed by placing a smaller tube within a larger tube, leaving a gap between the two.

Figures 5a and Sb show two probes that are equipped with metal conductors (60) that can be used for capacitive level-detection. (The conductors are connected via connections that are not shown in figures 5a and 5b to electric or electronic sensing circuits.) Advantageously, each conductor should have a relatively large surface area that is parallel to the surface of the sample, because this increases the capacitive coupling between the conductor (60) and the sample.

In use the probe is lowered until a change in its capacitance of a predetermined magnitude is detected.

This may coffespond to a known gap between the probe and the sample.

Such detection of the sample liquid level is particularly useful if the volume of the sample liquid is not known.

The probe can be constructed with polar, glass-like, metallic, non-polar, hydrophilic, hydrophobic, fluorinated or silanized surfaces. Hydrophobic surfaces (including fluorinated and silanized surfaces) reduce the tendency for aqueous materials to adhere to the probe, but they are prone to contamination by protein because protein is denatured by very hydrophobic surfaces. The surface an also be treated to prevent the denaturation of protein. For example, a surface with poly-ethylene glycol groups (a "PEG brush") would be have this property.

Claims

What I claim is: 1. A method of loading at least one sample into a conduit by way of a probe where steps are taken to ensure that an immiscible liquid remains between the probe and the sample.
2. A method as claimed in claim 1 comprising the steps of: (1) providing at least one sample liquid a receptacle, (I) covering each sample liquid with a covering liquid that is immiscible with it, (3) providing a probe in the form of a tube or member with a bore, (4) moving said probe so that its orifice is beneath the surface of said covering liquid and close to but not touching the surface of said sample liquid, (5) aspirating liquid into said probe sufficiently fast that both said sample liquid and said covering liquid are drawn into the bore of said probe.
3. A method as claimed in claim 1 comprising the steps of: (1) providing at least one sample liquid in a receptacle, (1) covering each sample liquid with a covering liquid that is immiscible with it, (3) providing a probe in the form of a tube or member with a bore, (4) moving said probe so that its tip is beneath the surface of said sample liquid, (5) aspirating liquid including sample liquid into said probe before the probe makes contact with the sample liquid, and (6) raising said probe above the surface of said sample liquid.
4. A method as claimed in claim 1 comprising the steps of: (1) providing at least one sample liquid in a receptacle, (1) covering each sample liquid with a covering liquid that is immiscible with it, (3) providing a probe that possesses a main bore for aspirating the sample liquid and at least one additional bore or orifice for ejecting said covering liquid, (4) moving said probe so that its tip is beneath the surface of said sample, (5) aspirating liquid including sample liquid into said probe, and (6) raising said probe above the surface of said sample liquid.
5. A probe suitable for carrying out the method of claim 1 that takes the form of a tube.
6. A probe suitable for carrying out the method of claim 1 that possesses an opening that is flared, chamfered or radiused.
7. A probe as claimed in any previous claim where a flange around the probe provides a flat or curved surface around the orifice of said probe.
8. A probe as claimed in claims 5 to 6 which is a thick-walled tube that provides a flat or curved surface around the orifice of said probe.
9. A probe as claimed in any previous claim that possesses a main bore and at least one additional bore or orifice that allows a covering liquid to be ejected close to the main bore.
10. A probe as claimed in any previous claim that possesses a conductor at or near the lowest part of said probe that can be used to detect the level of said sample in said receptacle.
11. A probe as claimed in any previous claim that possesses a surface that has polar, glass-like, metallic, non-polar, hydrophilic, hydrophobic, fluorinated or silanized surfaces.
12. A probe as claimed in any previous claim that possesses a surface that prevents the denaturation of protein.