Background Art Specimens and reagents in laboratories are now often dispensed in specific volumes using air displacing devices (pipettors) equipped with disposable plastic pipette tips. This makes for convenient, clean and reproducible dispensing of volumes of fluids. The use of disposable tips ensures no cross contamination and good reproducibility.

Microwell/microplate technology and the common use of immunoassay methods such as ELISA has meant that large numbers of specimens are often dispensed by an operator into an array of microwells. More recently robotics have started to automate such techniques.

Various proprietary solutions used in an ELISA, such as the sample diluent, the wash fluid, the conjugate and the substrate are now often colour coded to reduce any risk of performing the procedure incorrectly. However until now it has not been possible to do this for the very first step of such procedures which involves sample dilution. Thus it is difficult to check whether a particular test sample has actually been applied into a specific well containing dilution fluid. This is because the sample and dilution fluid may have similar protein composition, a similar appearance and the volume change may be very small. Thus one disadvantage of current ELISA procedures is that an operator may lose track of progress with specimen dilutions regardless of whether he or she is alert.

Sometimes a small, barely visible microclot can block a pipette tip and prevent sample pickup or delivery. Another disadvantage is that the operator may also lose concentration due to the repetitive nature of such large scale pipetting and again introduce errors into the procedure.

Disclosure of the Invention It is an object of this invention to overcome at least one of the above disadvantages with pipette tips currently in use.

Therefore, according to a first aspect of this invention there is provided a pipette tip having a pipette-body engaging end and a fluid receiving and delivery end, said pipette tip having a tracer coated on at least one surface selected from the inside surface thereof and the outside surface thereof and proximate the fluid receiving and delivery end, which tracer is capable of mixing with a liquid sample drawn into the pipette tip.

According to a second aspect of this invention, there is provided a method of coating or partially coating with a tracer, at least one surface selected from the inside surface thereof and the outside surface thereof, said pipette tip having a pipette-body engaging end and a fluid receiving and delivery end, which method comprises dissolving said tracer in a relatively volatile solvent to form a solution of said tracer and at least partially immersing the fluid receiving and delivery end of said pipette tip in said tracer solution; removing said pipette tip from said tracer solution and allowing the solvent to evaporate, thereby leaving a substantially even coating of said tracer on said at least one surface of said pipette tip.

Tips can be coated with different tracers on the inside and outside surfaces by temporarily blocking the opening in the tip and applying different tracer solutions to the external and internal surfaces. Additionally, tips can be coated either on the inside or the outside surfaces by temporarily blocking the opening in the tip and applying the tracer solution either to the external or internal surfaces.

The tracer may be any chemical, dye, pigment, marker or other tracer which can be coated over the surface of a pipette tip and then transferred from that pipette tip to the sample and subsequently be optically detected in the diluted sample can be used. It is preferred that the tracer coated on the pipette tip be inert in relation to the sample and the assay for which the sample is being diluted and tested.

For example, when,. the tracer is a dye it can be a pH indicator such as bromcresol purple which is yellow or colourless when applied to the pipette tip and can change or gain

colour to purple when added to the dilution fluid with neutral pH. Alternatively it can be any dye which is less coloured when dry than when wet. Most preferred dyes are bromcresol purple, bromphenol blue, tetrabromophenol blue, bromcresol green, chlorophenol red, bromthymol blue, phenol red, cresol red, m-cresol purple, thymol purple and thymolphthalein.

Other preferred tracers include, but are not limited to fluorescent dyes such as fluorescein, rhodamine and eosin, as well as regular stains crystal violet, rosaniline, Coomassie blue, Malachite green and Ponceau red. When the tracer is a fluorescent compound, it would require lower concentration than a dye and would be more visible under a UV light.

Commonly, the tracer is a dye in which case, the concentration of dye dissolved in the volatile solvent is suitably between about 0. 1g/L and about lOOg/L and is preferably between about lg/L and about lOg/L and more preferably about 4g/L.

The pipette tip is usually of the disposable type and is also usually plastic.

A pipette tip coated with tracer according to this invention may be prepared by applying a tracer with or without film-forming additives dissolved in a relatively volatile solvent.

The solvent is a relatively volatile solvent, and is more preferably a chlorinated hydrocarbon volatile solvent. The solvent may be for example, chloroform, ethanol, methanol, carbon disulphide, toluene, ether, ethyl acetate, formamide or any suitable volatile solvent known in the art such that after the tip is withdrawn from the tracer solution, the solvent readily evaporates off. The most preferred solvent is chloroform.

Processing with such potentially harmful solvents should be done in a closed manufacturing environment so that the solvent can be recovered, recycled and not released into the environment.

Aqueous solutions of tracers can be used provided that the pipette tip surface has been pretreated to allow even wetting with surface-bound agents such as polymers (eg. synthetic such as polyvinyl pyrrolidone and cellulose derivatives or natural such as proteins, albumin, gelatin, fibrinogen) and surface active agents (preferably non-ionic, such as Tween 20 or Triton X100) to allow even wetting.

The temperature at which the tracer is applied is ambient room temperature and is not particularly critical although a temperature which would soften the pipette tips should clearly be avoided.

Additives can include agents such as polymers such as polyethylene glycol or cellulose derivatives which assist in the formation of stable, thin, soluble films on the surface of the tip. In practice pipette tips are rinsed with this solution, drained and dried.

Although this invention may be used on a single tip, the advantages are more fully realised where pipette tips are moulded into a row of pipette tips, which commonly use up to 8 in a row or in a 96 tip array for use in assay procedures such as the ELISA technique and the like.

In use, samples such as patient sera are picked up by a pipette tip in the conventional way by dipping the tip into the sample and drawing up a volume of sample. A trace of indicator dye on the outside of the tip dissolves in what is left of the specimen to visibly mark that it has been accessed. More importantly, some dye from the inside of the pipette tip dissolves in the sample which has been picked up and is released with the volume mixed with the dilution fluid. The diluted sample acquires a visible colour, usually blue, and can be mixed appropriately until the colour is evenly distributed.

A pipette tip according to this invention is thus able to release the tracer into the sample, the tracer being carried along with the sample which can then be used to mark wells which have been loaded with the sample.

Again, as an example, a pipette tip coated with a dye can leave a visible trace of the dye in the remaining specimen volume when the dye is coated on the outside of the tip and can leave a visible trace of dye in the diluted sample when the dye is coated on the inside of the tip. If both the inside and outside surfaces are coated with the dye, then a visible trace of dye will be left in the specimen and diluted sample. This visible check provides assurance that any particular sample has been accessed and delivered.

A further advantage of the present invention is that a more precise check of optical density at the tracer's optimal wavelength can ensure that the correct volume of sample has been delivered. The amount of dye released from an evenly-coated pipette tip surface

is proportional to the surface area which has been contacted. Since this surface area is proportional to the sample volume picked up the optical density may be used to check volume delivery. This could be of benefit in a fully automated robotic process.

Tracer coated tips can be used to conveniently deliver marker dye or stains to a number of applications other than just in dispensing samples for ELISA. Thus they can be used for adding precise amounts of indicators and markers to test solutions. Indicator dyes such as bromcresol purple are still sometimes used for checking pH levels in test solutions and this can be done very conveniently in microtitre wells quantifiable in microplate readers.

Redox indicators such as methylene blue can also be added in precise and consistent quantities via pipette tips to test solutions to determine oxidation/reduction potentials.

Tracer coated tips can also be used for adding the traditional electrophoresis tracking dyes such as bromophenol blue to specimens being prepared for electrophoresis, thereby avoiding the use of a separate solution.

Brief Description of the Drawings Fig 1 is a representation of the pipette tip of this invention partially immersed in a solution containing a tracer.

Best and Other Modes for Carrying out the Invention Referring to Figure 1 pipette tip 1 has a pipette-body engaging end and a fluid receiving and delivery end 3. Pipette tip 1 has a tracer coated on at least one surface selected from inside surface 4 and outside surface 5 and proximate the fluid receiving and delivery end 3. The tracer is capable of mixing with a liquid sample drawn into pipette tip 1. Inside surface 4 and outside surface 5 of pipette tip 1 may be coated by partially immersion in a solution which contains a tracer dissolved in a relatively volatile solvent.

In use, samples such as patient sera are picked up by pipette tip 1 in the conventional way by dipping fluid receiving and delivery end 3 into the sample and drawing up a volume of sample. A trace of indicator dye on the inside surface 4 and/or outside surface 5 dissolves in what is left of the specimen to visibly mark that it has been accessed. More importantly, some dye from inside surface 4 dissolves in the sample which has been picked up and is released with the volume mixed with the dilution fluid. The diluted

sample acquires a visible colour, usually blue, and can be mixed appropriately until the colour is evenly distributed.

The present invention is illustrated by way of the following examples which are not to be construed as limiting on the scope thereof.

Example 1 Coating pipette tips with bromcresol purple Bromcresol purple (BCP; 5,5'-dibromo-o-cresolsulphonphthalein, sultone form, B-5880, Sigma-Aldrich, St Louis, USA) is dissolved at a concentration of about 4g/litre in chloroform in a glass container.

Pipette tips (eg from Eppendorf, Germany) suitable for 50pL-200uL volume dispensing, are dipped into this solution to a depth of about lcm. They are allowed to drain for about 30sec on blotting paper and then are blown dry with a fan dryer. This is most efficiently done with pipette tips arranged in a microplate array of 8 x12 = 96 tips.

Example 2 Use of bromcresol purple-coated tips in an assay Sera being tested for antibodies against dsDNA (ELISA for lab diagnosis of SLE) : A 5pL volume of patient serum is picked up with a Gilson pipettor equipped with a bromcresol purple-coated tip and transferred to a specific well in a dilution plate where each well contains 24511L of diluting fluid. The solution is mixed (pumped up and down with the pipettor) until the blue colour in the 1/50 dilution is evenly distributed. Then the tip is discarded and the process repeated for all sample sera. (Note that the rest of this ELISA is irrelevant to the coated pipette tip concept).

The 1/50 sample dilutions (100pL/well) are then transferred to corresponding wells in a dsDNA-coated microplate using a multichannel pipettor equipped with regular tips to start the ELISA proper. After an incubation of 30min, wells are drained, washed and filled with 100 lit of dilute HRP anti human IgG/A/M conjugate solution. After a further 30 min incubation, wells are again drained and washed several times. Then 1001lL of a stock HRP-sensitive substrate solution containing tetramethylbenzidene, buffers, stabilisers and hydrogen peroxide is added for 10min after which time the colour-

producing reaction is stopped with 100pL of 1M sulphuric acid. The optical density of all wells is read at 450nm on a microplate reader. Absorbances are converted to FARR units/mL by comparison against known references.

The use of bromcresol blue-coated tips in sample preparation for this ELISA has been shown not to affect the results for 48 random patients.

Industrial Applicability The present invention should find wide application in laboratories where disposable pipette tips are in common use such as pathology and analytical laboratories and the like.

Modifications obvious to those skilled in the art may be made thereto without departing from the spirit of the invention. For example, a leachable dye may be incorporated into the plastic used to manufacture pipette tips except that the amount needed would be much higher than if it were simply coated onto the surface. The tracer could also be included via a tracer mould-release agent which could be used during the plastic injection moulding process. Alternatively, the tracer solution could be sprayed onto or brushed over the pipette tips.