Abstract:
A pipette tip comprises a vessel for attachment to a pipette, wherein the vessel is prefilled with a reagent or chemical to permit an assay to be carried out in the pipette tip itself. The pipette comprises a body portion for aspirating a fluid sample into the pipette tip when attached thereto, the body portion comprising at least one light source, or at least one entry point for light from at least one light source, providing an optical path of light that passes through the sample in a direction essentially along the longitudinal axis of the pipette tip.

Description:
TECHNICAL FIELD 
       [0001]    The present invention relates to a pipette tip, as well as a pipette for use in spectrophotometrical analyses, and to an apparatus and a kit for the measurement of light absorbed by or emitted from a liquid sample. The present invention also relates to a method of using the pipette tip, pipette, apparatus and kit. 
       BACKGROUND ART 
       [0002]    In recent years a great deal of technology has been developed to handle small volumes of liquid samples. Pipettes are liquid handling tools that are commonly used in molecular biology as well as in medical tests. Conventional pipettes generally include an elongated cylindrical body having at one end a coaxially mounted pipette tip, a cylindrical piston within a cavity of the pipette body, and an actuating mechanism for actuating the piston. The actuating mechanism may cause the piston to perform an upward stroke so that liquid is aspirated into the pipette tip or a downward stroke so that liquid is dispensed from the pipette tip. 
         [0003]    In a wide range of fields, samples are analysed by measuring their light absorbance. Biological samples such as nucleic acids and proteins are analysed in this manner in the biotechnological field, for example. In a spectrophotometer, light having a known intensity at a variety of wavelengths is beamed at a sample, the light is detected after it has passed through the sample and is analysed for the absence, or reduced intensity levels, of certain wavelengths of light. This information, along with sample thickness, is used to identify and measure the concentration of substances in the sample. 
         [0004]    Samples that produce fluorescence or luminescence may be analysed by measuring their light output. Measurement of the intensity or life of fluorescence or luminescence emitted from a sample provides information on the physical or chemical properties of the sample. 
         [0005]    Biological samples may only be available in minute amounts for analysis and quantitative determination. For analysis in a light measuring apparatus, such as a spectrophotometer or fluorometer, the sample to be analysed is usually contained in a vessel referred to as a cell or cuvette, whose sides permit the passage of those wavelengths needed to characterize the sample contained therein. In an apparatus of the kind described, an optical beam of light generally enters the cuvette through one transparent end of the cuvette and exits the cuvette at the opposite end thereof. The characteristics of the beam emerging from the cuvette are then analysed to determine the composition of the fluid through which the light beam has passed and which is contained in the cuvette. Since the light beam has to pass through the cuvette, the transparent material thereof may cause inaccuracies in the determination of the composition of the fluid. 
         [0006]    Further, due to the minute size of the samples analysed in the apparatus described, problems arise such as loss of sample due to transfer from pipette to cuvette, sample volume being too small for the size of the cuvette, evaporation of sample during analysis, recovery of sample after analysis, and contamination of sample after recovery. 
         [0007]    Moreover, when a sample to be analysed has a high density, additional measures may need to be taken to reduce sample density prior to light measurement so that sufficient light from the light source can pass through the sample for detection by the photo detector. An example of a known measure to reduce sample density is dilution of the sample prior to light measurement. On the other hand, when a sample has a low density, the intensity of light passing through the sample from the light source may be too high for measurement by the photo detector. 
         [0008]    There is therefore a need to increase the accuracy of spectrophotometrical analyses done on fluids, especially liquids, such as pure liquids, solutions, dispersions, colloidal solutions or the like, particularly on minute volumes of liquids. 
       SUMMARY OF THE INVENTION 
       [0009]    The present invention provides a pipette tip, pipette, an apparatus and a kit for use in the spectrophotometrical analysis of a fluid sample, and a method for use of the same, in which the above mentioned problems associated with the analysis and quantification of minute amounts of fluid sample are overcome. 
         [0010]    In accordance with a first aspect, the present invention provides a pipette tip which comprises a vessel for attachment to a pipette, wherein the vessel is prefilled with a reagent or chemical to permit an assay to be carried out in the pipette tip itself and wherein the pipette comprises a body portion for aspirating a fluid sample into the pipette tip when attached thereto, the body portion comprising at least one light source, or at least one entry point for light from at least one light source, providing an optical path of light that passes through the sample in a direction essentially along the longitudinal axis of the pipette tip. Preferably, the body portion is additionally adapted for discharging fluid through the intake or an opening defined by the pipette tip distal to the pipette. 
         [0011]    The use of the pre filled pipette tips for diagnostic purposes greatly facilitates near patient monitoring and simplifies diagnostics in general. 
         [0012]    In this regard, the invention is based on the pre-filling of the pipette tips with one or more reagents, so that a user can use a tip pre-filled with the reagents necessary to do e.g. a hepatitis elisa test. A fixed wavelength light in the pipette could then be used with a detector to measure absorbance. 
         [0013]    A major benefit of the invention is the ability to use fluorescent detection. Thus, in accordance with the invention, it is possible to shine light of one colour (wavelength) down the pipette into one or more reagents in the pipette tip, which could also contain a fluorescent label. If a sample taken into the pipette tip reacts with the reagent(s) to give a positive result (or negative or whatever result a user is looking for), when it mixes with the reagent(s) fluorescence occurs which would be at a different wavelength from that of the incident light and hence distinguishable as being from due to the presence of a reactant in the sample. 
         [0014]    Further advantages of the invention include the pre-filling of one or more reagents of interest that are particularly difficult to pipette accurately. 
         [0015]    In addition, the invention assists in avoiding the risk of contamination, oxidation or aeration of reagents, which can occur when a reagent is drawn up into a pipette tip by a pipette. 
         [0016]    In accordance with the present invention, a reagent is placed in the pipette tip prior to use. By leaving the reagent in the pipette tip for a prolonged time any aeration caused by the pipetting is reduced. This increases accuracy of the amount of reagent in the pipette tip and assists with accurate spectroscopic testing of a reagent and sample in the pipette tip. 
         [0017]    Pre loaded tips provide the advantage of ensuring that the correct volumes of the correct reagents are used in an assay. This prevents a user in a lab from making a mistake with e.g. the volume, concentration or specification of reagents. Absolutely removing the possibility of user error is of great importance in ensuring that results of an assay are valid. By pre loading the tips with defined reagents it removes a possible source of variability from the tests. 
         [0018]    In one embodiment, the invention provides a method wherein a sample is drawn into a pipette tip in a number of steps, shining a light longitudinally through the pipette tip during the step process to obtain a calibration curve of the light detected. 
         [0019]    A number of readings are taken as the liquid column is drawn into the pipette tip. Since each reading takes place through a column of different length, the optical density changes with each reading so that it is possible to produce a calibration curve for each set of readings, i.e. each time the column is drawn into the pipette tip. 
         [0020]    In essence, a small liquid sample is placed on a pedestal below which is a light detector. The pedestal transmits UV as in a UV/Vis spectrophotometer. The end of the pipette tip is placed on the sample, and a minute amount to the sample is aspirated into the pipette tip in a number of steps, while light is transmitted through the sample from the pipette into the detector below. This enables the pipette to be used to quantify e.g. DNA or protein concentration. 
         [0021]    Preferably the number of steps is about 1 to about 10. More preferably, the number of steps is about 4 or 5. Most preferably, the number of steps is at least 4. 
         [0022]    Liquid handling is a fundamental part of sample preparation for most assays. Many assays have an end point, which is measured spectrophotometrically. Historically, pipettes and automated liquid handling devices have been used to transfer reagents into a suitable vessel, e.g. a microplate well or quartz lens, through which light can be passed to qualify or quantify absorbance/fluorescence/luminescence and hence the contents of the sample. 
         [0023]    Electronic pipettes in particular have a power source. They tend to be held in stands, which have a charger and a base. If, for example, a light detector is placed in the base and a light source is placed inside the pipette, then the pipette can be used for a complete assaying process. Thus, for example, in an Elisa assay, a known volume of sample can be aspirated into a pipette tip and the liquid travels a known distance up the pipette tip, as calculated by the internal diameter of the pipette tip and the volume of sample being aspirated. The sample can then be ejected, leaving e.g. antibodies or antigens, which were present within the sample coated on the internal surface of the pipette tip. The area being coated would be known by the length of the liquid column drawn into the pipette and the internal diameter of the tip. In Elisa assays, once sample is adsorbed onto the internal surface of the tip, excess unbound sample can be removed by aspirating wash buffer up and down the tip. Ultimately the substrate can be aspirated into the tip. Absorbance of light of appropriate wavelength (from the light source inside the pipette) is then measured by the detector and the sample identified and quantified, according to established principles, in this case of Elisa. 
         [0024]    Pipette tips of different materials, which adsorb and retain different reagents at different or optimal levels can be used. 
         [0025]    Ultimately, drawing a long liquid column into a pipette tip and facilitating reactions over a consequently relatively large surface area, then reading the result through a long liquid path (optimal according to Beer&#39;s law) with no interface caused by a container between the light source and the detector, optimises results and minimises noise so that the signal to noise ratio is maximised. 
         [0026]    According to the invention, there is no chance of cross contamination. Reagents are completely contained with the pipette tip and each reaction occurs in a new pipette tip. Hence there is no opportunity for cross contamination, which is very important in many assays. 
         [0027]    A further example of an assay, which can be carried out according to the invention, is DNA quantification. Again, due to the narrow internal diameter of the pipette tip, one or two microliters of DNA will form a relatively long column within the pipette tip. UV light from a light source inside the pipette travels through the sample into the detector. 
         [0028]    In one embodiment the pipette tip has an opaque wall to prevent light from escaping or entering from outside. However light travels through the sample in the pipette tip due to internal reflection within the liquid. 
         [0029]    The ability to carry out assays in a pipette tip opens up many further opportunities. In this regard, in one embodiment, pre coated tips and a range of custom “peripherals” and disposables make it easier for the operator to carry out the assays. 
         [0030]    Pipettes capable of being used to carry out complete assays rather than just the liquid handling in a pipette are advantageous. In this regard, an entire assay can be carried out in a pipette tip rather than merely using pipettes for liquid handling. Traditional assays can be switched to the “test in a tip” format. 
         [0031]    A pipette according to the present invention may include the pipette tip attached to the body portion. The light source is preferably provided within the pipette body. When the light source is provided remote from the pipette body, preferably light from the light source is guided by means of, for example, an optical cable or fibre to the open end of the pipette tip that is attached to the pipette body so that the light passes through the inside of the pipette tip. 
         [0032]    The pipette tip may be detachably retained on the body portion. In this way, the pipette tip may be removable from the body portion for disposal and replacement. Alternatively, the pipette tip may be integral with the body portion and may take the form of a pipette probe having an integral tip that is washed for re-use between sample collections to remove any contamination. 
         [0033]    In accordance with the present invention there is provided a method for measuring light output from a fluid sample that produces luminescence, comprising providing a pipette tip or capillary tube having first and second open ends with the sample to be analysed contained therein, and detecting light output from at least one open end of the pipette tip or capillary tube. 
         [0034]    When the fluid sample is one that when irradiated with light absorbs the light or fluoresces, the method preferably further comprises providing a light source, permitting light from the light source to enter one open end of the pipette tip or capillary tube, to pass through the sample contained therein, and to leave the pipette tip or capillary tube by its other open end for detection and analysis. The pipette tip or capillary tube is elongate and the path of light preferably passes through the sample contained in the pipette tip or capillary tube in a direction essentially along the longitudinal axis of the pipette tip or capillary tube. 
         [0035]    The method according to the present invention preferably further comprises determining light emission or absorbance of the sample according to the intensity of the light detected. 
         [0036]    In the method according to the present invention, the pipette tip is preferably retained on a pipette body during sample analysis and the method, optionally, further comprises recharging the pipette body during sample analysis. In a preferred method, the light source is provided within the pipette body. 
         [0037]    The method according to the present invention preferably further comprises controlling the light input to the sample, for example the intensity and/or wavelength of light input to the sample. 
         [0038]    In accordance with the present invention there is provided an apparatus for light measurement from a fluid sample, wherein the apparatus comprises a container for the sample in the form of a pipette tip or capillary tube having first and second open ends, and a photo detector for detecting light output from the sample, wherein the photo detector and the pipette tip or capillary tube are disposed so that light output from the first and/or second open end of the pipette tip or capillary tube is detected by the photo detector. 
         [0039]    Preferably, the apparatus further comprises at least one light source, wherein the at least one light source is disposed to input light through one open end of the pipette tip or capillary tube so that the light passes through the sample contained therein, and wherein the photo detector is disposed to detect light output from the other open end of the pipette tip or capillary tube. The optical path of light from the light source preferably passes through the sample in a direction essentially along the longitudinal axis of the pipette tip or capillary tube. 
         [0040]    More preferably, the apparatus further comprises light output guide means for guiding light output from a first open end of the pipette tip or capillary tube to the photo detector and/or light input guide means for guiding light from the light source to a second open end of the pipette tip or capillary tube. The light input and/or output guide means may take the form of an optical fibre or cable. 
         [0041]    The apparatus may further include a pipette body, wherein the pipette tip of the invention is attached to the pipette body, such as being integral with, or detachably retained on, the pipette body. 
         [0042]    In the apparatus according to the present invention, the at least one light source may be disposed within the pipette body, or the pipette body may be provided with at least one entry point for light from the at least one light source. Ideally, a passageway is provided within the pipette body for directing light from the light source to the inside of the pipette tip and the passageway is preferably coated with a light reflective material. A piston may be provided within the pipette body and the light source may be attached to the piston. In one embodiment, the piston has an inner space and the light source is housed within the inner space, where, optionally, the walls of the inner space of the piston are coated with a light reflective material. In another embodiment, the piston is provided with an axial bore or optical fibre for guiding light from the light source to the pipette tip. The light source is preferably disposed on the longitudinal axis of the pipette body. 
         [0043]    The apparatus according to the present invention preferably further comprises a holder for holding the pipette tip or capillary tube in position during light measurement. The holder may be adapted to hold a plurality of pipette tips and/or capillary tubes for simultaneous detection of light output from multiple samples. It may take the form of a rack or a plate with a plurality of apertures, for example. The holder may be adapted to hold a pipette body with attached pipette tip. The holder is preferably adapted to hold the pipette tip so that it is disposed in a generally horizontal plane. In this way, leakage of sample from the open ends of the pipette tip is avoided. Preferably, however, the diameters of the first and second openings of the pipette tip are such that the liquid sample is retained therein by means of its surface tension so there will be no leakage from the pipette tip even when positioned in a generally vertical plane. 
         [0044]    The pipette tip of the invention may be mounted in the holder by the pipette body or probe with attached tip being manipulated manually or by means of a robotic arm. A frictional fit between the pipette tip and the holder may be provided such that the pipette tip with sample contained therein will remain on the holder when the pipette body is subsequently moved away. In one embodiment, the holder for the pipette tip is removable from the light measuring apparatus for loading and unloading purposes. 
         [0045]    The apparatus according to the present invention may further comprise a fluid-tight housing for at least the pipette tip or capillary tube, wherein the inner space of the housing is in communication with the internal space of the pipette tip or capillary tube in use. In this way, interference from outside radiation is reduced. The light source may be disposed within the internal space of the apparatus. 
         [0046]    In the case when the pipette body portion remains attached to the pipette tip during light measurement, the fluid tight housing of the apparatus includes an attachment portion for the pipette to allow the pipette to be in the correct position for sample analysis. One or more detents may be provided on the exterior surface of the pipette body portion for engagement with one or more indentations of the attachment portion. For example, the pipette body portion may be provided with a bayonet and the attachment portion may comprise a bayonet socket. In this manner, the pipette can be easily attached and detached from the apparatus. By having a holder or attachment portion, the pipette tip is held firmly in the measuring position so that misalignment of the optical axis of the optical path of light is reduced. 
         [0047]    Preferably, the apparatus according to the present invention further comprises means for recharging the pipette body during light measurement. The light source in the pipette body may require recharging and/or the pipette may be an electronic pipette that requires recharging. The attachment portion or holder of the apparatus and the pipette according to the present invention may be provided with electrical connections in accordance with known methods to allow for recharging of the electronic pipette and/or the light source. 
         [0048]    The apparatus or pipette according to the present invention preferably further comprise first control means for controlling the intensity of light entering the pipette tip and/or second control means for controlling the wavelength of light entering the pipette tip. 
         [0049]    The apparatus or pipette according to the present invention preferably comprises a plurality of light sources, whereby each light source is disposed to irradiate light of a predetermined intensity and/or wavelength. 
         [0050]    In accordance with the present invention there is provided a kit for light measurement, the kit comprising a pipette tip or capillary tube for containing a fluid sample to be analysed, the pipette tip or capillary tube having first and second open ends, and light output guide means for guiding light output from at least one open end of the pipette tip or capillary tube to a photo detector. 
         [0051]    The kit may further comprise light input guide means for guiding light from a light source to one open end of the pipette tip or capillary tube so that the light passes through the sample and leaves by the other open end of the pipette tip or capillary tube. 
         [0052]    An alternative kit according to the present invention comprises a pipette body portion for aspirating a fluid sample into a pipette tip when attached thereto, wherein the body portion comprises at least one light source, or at least one entry point for light from at least one light source, providing an optical path of light that passes through the sample in a direction essentially along the longitudinal axis of the pipette tip, and light output guide means for guiding light output from an open end of the pipette tip to a photo detector. The kit may further include one or more pipette tips for attachment to the pipette body portion. Preferably, the pipette body includes light source control means whereby the intensity of light (illuminance) is adjustable as required. 
         [0053]    The light input and/or light output guide means may take the form of an optical fibre or cable. 
         [0054]    In accordance with the present invention there is provided an apparatus for light measurement from a fluid sample, the apparatus comprising a container for the sample to be analysed, at least one light source for irradiating the sample with light, a detector for detecting light output from the sample, and a light source control for controlling the intensity of light input to the sample. 
         [0055]    In the apparatus according to the present invention, the light source control may control light intensity in a number of different ways. For example, the light source control may control light intensity by varying the electrical power supplied to the light source. The electrical power may be varied between no power (OFF) and maximum power, with degrees of electrical power being selectable between OFF and maximum power. The light source may be implemented, for example, by a single Light Emitting Diode (LED) or by an array of LEDs to provide illumination. By adjusting the electrical power to one or more of the LEDs in the array, for example, by turning off one or more of the LEDs, light intensity may be reduced. The light source control may be a dimmer switch, for example, the operation of which is well known. In one embodiment, the distance of the light source relative to the sample may be adjusted to control the intensity of light beamed at a sample. In this regard, the light source may be movable towards or away from the sample to permit variation of the optical path length and thereby the intensity of light beamed at the sample. In another embodiment, a screen may be disposed between the sample and the light source having a variable aperture and the light source control may control light intensity by varying the size of the aperture in the screen. For example, the aperture may be constructed of a number of blades that can close down to form a smaller aperture or completely open to form the maximum aperture. In a yet further embodiment, a shutter may be disposed between the sample and the light source and the light source control may control light intensity by opening the shutter for a predetermined period of time. The aperture and shutter construction may be similar to that provided in a camera. 
         [0056]    It is advantageous to be able to control light intensity. If the absorbance of the sample is high, that is, it has a high optical density, light of a high intensity is likely to be detected by the photo detector, whereas light of a low intensity is unlikely to be able to penetrate the sample and therefore may not be detected. Conversely, if the sample has a low optical density, light of a high intensity is likely to be out of range for the photo detector and therefore will not be detected, whereas light of lower intensity is likely to be detected and thereby will provide a quantifiable signal. Accordingly, by permitting the intensity of light from the light source to be adjustable, the light emitted from the sample may be controlled to be at a level that can be measured or is within the dynamic range of the photo detector. 
         [0057]    The light source control may be used in any optical measuring system where it would be desirable to be able to control light intensity, including micro titre plate readers, for example. 
         [0058]    The apparatus, pipette or kit according to the present invention may comprise a plurality of light sources and the intensity of each light source may be independently controllable. 
         [0059]    A single sample may be irradiated with multiple light beams. Alternatively, a plurality of samples may be irradiated with respective light beams of different wavelengths or intensities, simultaneously. 
         [0060]    The apparatus, kit or pipette body according to the present invention may comprise multiple input light guides for guiding light from one or more light sources to the pipette tip(s) and/or multiple output light guides for guiding multiple light beams output from the pipette tip(s) to respective multiple detectors. In an alternative embodiment, light from a single light source is divided into two or more light beams where each beam is directed to a different pipette tip. 
         [0061]    The apparatus according to the fifth aspect may be a spectrophotometer having a light source control as described herein for controlling the intensity of light input to the sample. 
         [0062]    The term “pipette tip” as used herein, is intended to encompass all types of pipette tips, including pipette tips used for automated and manual pipetting, positive displacement pipettes, the pipette tip may be integral with a pipette body, such as a pipette probe, pipette tips in the form of a capillary tube or pipette tips having a tapered inner passageway, pipette tips that are circular or flat in cross-section, and all other pipette tips. In general, the pipette tip has a hollow body which defines an interior volume and a channel therein extending from an intake opening to an attachment opening. Preferably, at least the intake opening of the pipette tip is of a diameter such that a liquid sample will be retained inside the pipette tip by means of its surface tension. The volume of the pipette tip may be in the range of 1 μm to 5 ml, typically greater than zero to 200 μm. Various plastics, e.g., polypropylene, silica, make ideal pipette tip materials as is well known in the art. 
         [0063]    The “first open end” or “intake opening” of the pipette tip as used herein is the end from which a predetermined amount of liquid sample is aspirated and may be dispensed. The “second open end” or “attachment opening” of the pipette tip as used herein is the end that is adapted to engage with a pipette body. The pipette tip may be mounted on a pipette body by means of a frictional fit between coacting surfaces on the pipette tip and the pipette body, for example. 
         [0064]    Preferably, the pipette tip for use in the apparatus or kit, or with the pipette body, in accordance with the present invention has substantially parallel inner walls in cross section taken along a central axis. The pipette tip preferably has a relatively long fine inner bore, like a capillary tube, so that the length of the path of light through the sample is relatively long even when the amount of the sample is minute. In this way, light passes through the maximum length of sample. 
         [0065]    The term “pipette body”, or “body portion” when used in relation to a pipette, is intended to include any fluid handling device that is capable of aspirating (i.e., drawing) a fluid into a column (pipette tip) attached thereto and, optionally, capable of discharging (i.e., expelling) fluid out of the column. 
         [0066]    The pipette according to the present invention may be of the type having an elongated cylindrical body with a coaxially mounted pipette tip at one end, a cylindrical piston within a cavity of the pipette body, and an actuating mechanism for actuating the piston. 
         [0067]    By use of a pipette tip as the container for both the collection and analysis of a sample, it is possible to accurately analyse small volumes of sample. In this way, dilution of a sample is not necessary and the problems mentioned above such as loss of sample through transfer from pipette tip to cuvette, evaporation of sample during analysis, reduced recovery and contamination of sample, are avoided. 
         [0068]    According to the present invention, light irradiated at a fluid sample from a light source takes the longest possible path through the sample in the pipette tip. Thus, by use of the pipette tip as the container for sample analysis and by use of an optical path of light that passes in a direction essentially along the longitudinal axis of the pipette tip, the light takes a long path through the sample relative to the small volume of sample available for analysis resulting in an accurate measurement of the sample. Further, because light from the light source passes through the open ends of the pipette tip, it passes through only the sample, without having to pass through the material of the pipette tip, resulting in analyses that are independent of the material of the sample container. 
         [0069]    The apparatus according to the present invention preferably further comprises means for measuring the length of sample column within the pipette tip. The means for measuring the length of sample column may comprise a digital camera. 
         [0070]    When light is absorbed by the sample, the reduction of light transmission by means of sample volume relative to a control sample is determined, and when light is emitted from the sample, the increase in light emission by means of sample volume relative to a control sample is determined. The volume of sample may be calculated automatically from a determination of length of sample column and internal diameter of pipette tip. The pipette tip may include a scale for use in measuring the length of sample column within the tip. 
         [0071]    Preferably, the photo detector is capable of detecting light intensities of a plurality of components having different wavelengths from the light output from the sample. The sensitivity of the photo detector can preferably be varied to permit the measurement of samples with a wide range of optical densities or wide range of optical emission intensities. 
         [0072]    Preferably, the light measuring apparatus according to the present invention further comprises a receptacle for collecting excess sample dispensed from the pipette tip prior to light measurement or for collecting sample dispensed from the pipette tip after light measurement for re-use or disposal. The receptacle may be used, for example, to collect sample released from the pipette tip when it is desirable to reduce sample path length, such as when the sample is of high optical density. The receptacle may be axially moveable relative to the pipette tip when held in position to enable sample to be collected from pipette tips of different lengths. 
         [0073]    Preferably, the light measuring apparatus according to the present invention comprises at least one lens for focussing light outputted from the sample to the photo detector. The pipette preferably comprises alternatively or additionally at least one lens disposed between the light source and the pipette tip for focussing light emitted from the light source into the pipette tip. 
         [0074]    The pipette, apparatus or kit according to the present invention may be calibrated by passing a range of predetermined test light intensities or wavelengths from the light source through the pipette tip when there is either no sample or a predefined control or calibration sample (e.g. distilled water or other solvent for the specimen to be tested) in the pipette tip. The intensity or wavelength of the test light emitted from the pipette tip containing no sample or a control or calibration sample is detected by the photo detector to provide a reference value. 
         [0075]    The photo detector outputs a signal corresponding to the intensity or wavelength of the light received from the sample. A change in light intensity is determined according to conventional methods. In one method, the signal output from the photo detector may be converted to a voltage signal and the voltage signal is fed to a computer which determines the light intensity corresponding to the voltage signal. 
         [0076]    The apparatus according to the present invention preferably further comprises means for measuring the length of sample column within the pipette tip or capillary tube, optionally wherein the means comprises a digital camera. 
         [0077]    The method according to the invention may accordingly include determining the length of the sample optical path. Sample optical path length may be calculated in a number of ways. It may be possible to visually measure path length by use of a separate scale or the pipette tip may be provided with a scale corresponding to the optical path length. Further, a camera may be used to determine or confirm the optical path length. The sample optical path length can also be determined from knowledge of the kind and form of the employed pipette tip and the amount of sample. A pipette can withdraw a known volume of sample, for example 1 μl, and knowledge of the internal diameter or dimensions of the pipette tip permits the path length to be calculated. The information can be stored into a computer beforehand so that the path length can be easily determined. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0078]      FIG. 1  is a longitudinal sectional view showing one embodiment of an apparatus in accordance with the present invention; 
           [0079]      FIG. 2  is a longitudinal sectional view showing a pipette tip and pipette in accordance with the present invention; 
           [0080]      FIG. 3  is a cross-sectional view taken along the line A-A′ of  FIG. 2 ; and 
           [0081]      FIG. 4  is a schematic representation of an alternative embodiment of an apparatus in accordance with the present invention. 
       
    
    
       [0082]    Preferred embodiments of this invention are described herein, including the best mode known to the inventor for carrying out the invention. It should be understood that the illustrated embodiments are exemplary only, and should not be taken as limiting the scope of the invention. 
       DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       [0083]    In the following, embodiments of the present invention will be described in detail with reference to the accompanying drawings, wherein like reference numerals represent like parts and assemblies throughout the several views. 
         [0084]      FIG. 1  illustrates an apparatus for light measurement comprising a light detector  48  within a housing  10 . A pipette, generally shown as  12 , is attached to the housing  10  and comprises a pipette body  14  and a pipette tip  16 . The pipette tip  16  and the distal end portion of the pipette body  14  are positioned within the inner space  38  of the housing  10  in the measuring position. The pipette body  14  includes a plunger mechanism comprising a piston  18  and a plunger button  20 . Attached to one end of the piston  18  is an annular ring  22  that abuts the inner wall of the pipette body  14  to locate the piston  18  centrally within the pipette body  14  and provide an air tight seal. At the other end of the piston  18  is provided a further annular ring  24  that is fixed to the inner wall  14 A of the pipette body  14  and allows the piston  18  to reciprocate back and forth therethrough. The piston  18  is hollow and a light source  26  in the form of a filament bulb is located within the hollow space  28  at the proximal end portion of the pipette body, near the plunger button  20 . The filament bulb  26  is fixed in position within the hollow piston. In another embodiment, however, the filament bulb  26  may be movable axially along the hollow space  28  of the piston  18  to be closer to, or further away from, the pipette tip  16  thereby, respectively, increasing or decreasing the intensity of light input to the pipette tip  16 , as required according to the density of the sample  36 . A lens  30  is fixed at the distal end of the piston  18  that seals the hollow space  28 . Light from the light source  26  is directed axially through the hollow space  28  within the piston body towards the pipette tip  16 . After passing through the lens  30 , parallel light is input to the sample  36  in the pipette tip  16 . 
         [0085]    The pipette tip  16  is pre-filled with reagent  36  and comprises a first open end  46  for transferring a liquid sample into and out of the pipette tip  16  depending on the magnitude of the pressure generated inside the pipette tip  16 , and a second open end  68  for attachment to the pipette body  14 . The pipette tip  16  comprises an upper section  17  that tapers downwardly to a body section  34  that, in  FIGS. 1 and 2 , is in the form of a capillary tube. The distal end of the pipette body  14  is configured and dimensioned for axial insertion into the second open end  68  of the pipette tip  16  to establish an axially interengaged relationship between the coacting surfaces of the distal end of the pipette body  14  and the upper section  17  of the pipette tip  16  so that the pipette tip  16  is detachably retained on the pipette body  14 . The reagent  36  for analysis is held in the inner passageway in the body section  34  of the pipette tip  16 . 
         [0086]    The pipette tip  16  and distal end portion of the pipette body  14  are located within the inner space  38  of the housing  10  for analysis of the reagent  36  held in the pipette tip  16 . The housing  10  has a pipette attachment portion  40  in one side wall  42  for receiving the pipette body  14 . The pipette body  14  is provided on its outside wall  14 B with a fixing in the form of two detents  42 ,  44  for engaging with respective recesses (not shown) provided in the inner wall  43  of the pipette attachment portion  40 . When in position attached to the housing  10 , the suction port  46  of the pipette tip  16  faces a light detector  48 , and a lens  50  is located between the light detector  48  and the suction port  46  for focussing light output from the suction port  46  onto the light detector  48 . 
         [0087]    A receptacle  52  is provided below the suction port  46  of the tip  16  for collecting any excess reagent  36  or any sample that after analysis is to be disposed of or retained and stored for re-use. The receptacle  52  is adapted to be movable axially relative to the pipette  12  so that it can collect samples released from pipette tips of different lengths. A digital camera  54  is provided on the outside of the spectrophotometer  10  having its lens  56  pointing through an aperture  58  in the wall  60  of the apparatus housing. The camera  54  is linked to a computer (not shown) and can be used to determine or confirm the length of the reagent column  36  in the body portion  34  of the pipette tip  16 . Camera exposure may be synchronised with absorbance measurement. 
         [0088]      FIG. 2  illustrates an alternative pipette  12  in accordance with the present invention. The pipette  12  is similar to the pipette  12  illustrated in  FIG. 1 , except that the piston  18  has an optic fibre  62  that passes through an axial bore  64  in the piston  18  (see also  FIG. 3 ) to direct light from the light source  26  to the pipette tip  16 . The lens  30  at the distal end of the piston  18  converts the light outputted from the end  62 A of the optical fibre  62  into parallel light, which is directed towards the suction port  46  of the pipette tip  16  in a direction along the longitudinal axis of the pipette tip  16 . 
         [0089]    Depression of the plunger button  20  under finger pressure against the tension of a spring (not shown) causes delivery of liquid reagent  36  from the capillary tube portion  34  of the pipette tip  16 . By permitting the tension of the coil spring to reverse the direction of movement of the piston  18  and plunger button  20 , liquid, for example a sample for analysis, is drawn into the capillary tube portion  34 . Reagent and sample optical path length is determined from the amount of the sample and reagent  36  contained in the tip  16 . The amount of sample and reagent  36  contained in the pipette tip  16  may be determined from the length of the sample and reagent column in the tip  16  and a knowledge of the bore diameter in the tip  16 . The amount of sample and reagent in the tip  16  may also be known from the setting on the pipette  12 . 
         [0090]    When a second sample is to be analysed, the pipette tip  16  is either washed or replaced. The first sample and reagent  36  is dispensed from the tip  16  and collected in the receptacle  52  by depression of the plunger button  20 . The receptacle  52  containing the sample and reagent may be stored, for example, so that the sample can be subjected to further tests, or washed for re-use. Alternatively, the receptacle  52  may be disposed of and replaced. 
         [0091]      FIG. 4  illustrates an apparatus comprising a pipette  12 , shown partially cut away, which is linked to a light detector  48  by means of a fibre optic cable  66 . The pipette  12  comprises a pipette body  14  and a pipette tip  16 , and within the distal end portion of the pipette body  14  is an LED light source  26  that beams light towards the pipette tip  16 . A dimmer switch (not shown) is provided to adjust the intensity of light emitted from the light source  26 , according to sample density. The pipette tip  16  has an opaque wall and comprises a first open end  46  for transferring a liquid sample and reagent  36  into and out of the pipette tip  16  depending on the magnitude of the pressure generated inside the pipette tip  16 , and a second open end  68  for attachment to the pipette body  14 . The pipette tip  16  comprises an upper section  17  and a body section  34  leading from the upper section and tapering downwardly to a reduced diameter first open end  46 . The surface of the distal end of the pipette body  14  acts against the surface of the upper section of the pipette tip  16  to provide a frictional fit between coacting surfaces. The pipette tip  16  has a tapered inner passageway  35  and is prefilled with a reagent  36  for analysis is held in the inner passageway  35  in the body section  34  of the pipette tip  16 . 
         [0092]    The light detector  48  is provided within a housing  10  in a fluid-tight environment. A fibre optic cable  66  connects the first open end  46  of the pipette tip  16  to the photo detector  48 . With the liquid sample  36  held in the pipette tip  16 , light from the light source  26  is input to the second open end  68  of the pipette tip  16  and passes through the whole volume of the sample  36  in a direction along the longitudinal axis of the pipette tip  16  (direction is shown with dashed lines), and any emitted light exits through the first open end  46 . The light that exits the first open end  46  is guided by the fibre optic cable  66  to the light detector  48 . An electrical signal proportional to the light detected by the light detector  48  is generated and analysed for determining quantitative or qualitative characteristics of the pipetted sample  36 . 
         [0093]    In an alternative embodiment, the light source  26  in  FIG. 4  may be provided within the housing  10  for the detector  48  instead of within the pipette body  14  and optical guide means, such as an optical fibre, may be used to direct light output from the light source  26  to the pipette tip  16 . 
         [0094]    In a further alternative embodiment, the pipette body  14  in  FIG. 4  may be absent and the light source  26  may be disposed to input light either directly or indirectly, for example via an optical fibre or cable, into the second open end  68  of the pipette tip  16  so that the light passes through the sample  36  in a direction along the longitudinal axis of the pipette tip  16 . 
         [0095]    The light source  26  may be a laser, LED, traditional filament bulb, or other light source. The light source may produce entirely visible light or light at least mainly at the infrared or ultraviolet range or a given waveband thereof. 
         [0096]    The specific embodiments described above are for analysis of a sample that will absorb light that is beamed through it. When the sample comprises a fluorescent material, a fluorometer is employed, wherein light, usually ultraviolet light, from the light source that is beamed through the sample causes the sample to emit light of a different energy or wavelength, typically visible light, and the emitted light is detected by a detector. When a luminescent sample is used, light is emitted from the sample for detection by the light detector and a separate light source is not required. 
         [0097]    The use of the terms “a” and “an” and “the” and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. 
         [0098]    All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. 
         [0099]    Although the specific embodiments described above relate to manual pipettes, the present invention is also applicable to automatic pipettes in which the piston is moved electronically according to input instructions. Indeed, it will be apparent to the skilled person that the present invention may be applied to various different kinds of pipettes, from those having pipette tips that can be discarded after use to those having probe tips provided with non retentive coatings, such as Teflon®, as used in robotic sample processors, for example. 
         [0100]    Further, the embodiments described above relate to single channel pipettes but the invention is equally applicable to multi-channel pipettes. It would be desirable to use multi-channel pipettes in a high-throughput screening method, for example. The absorbance measuring apparatus according to the invention would in this case be modified to have a plurality of pipette attachment portions and a plurality of photodetectors.