The present invention relates to an apparatus for analysing a sample, in particular to an apparatus for analysing proteins by measuring intrinsic fluorescence.

Proteins are composed of a variety of amino acids and of these, tryptophan and tyrosine absorb UV light at around 275nm. These amino acids then emit fluorescence typically at wavelengths above 300nm which can be detected. The amount and wavelength of fluorescence emitted correlates with the amount of tryptophan and tyrosine exposed to the surrounding solvent. This allows the degree to which proteins have unfolded to be detected and analysed by measuring the spectrum of intrinsic fluorescence of a protein sample. This can be done, for example, by using a spectrometer.

The amount of fluorescence of a protein can depend upon many factors. For example, a protein with a higher tryptophan and tyrosine content will emit more than a protein with a lower tryptophan and tyrosine content as will a protein at higher concentration. However, the shift in the peak wavelength (typically towards red wavelengths) is intensity independent and can be used as a reliable indicator of the transition of a protein from its natural conformation to an unfolded or denatured state.

These factors, therefore, mean that intrinsic fluorescence of proteins can be a very useful technique to analyse protein samples. In one example, in view of the effect on fluorescence by protein structure, denaturation of a protein from a folded state to an unfolded state can be analysed. In one example, known as thermal denaturation, the protein is provided in a solvent and then heated to unfold the protein. The shift in spectrum can be used to determine where the protein transitions from an unfolded to a folded state.

It is also possible to denature a protein, in particular an antibody, using chemical methods such as urea or guanidine hydrochloride. This technique is often known as chemical melt and can be combined with thermal denaturation to provide more accurate results.

These techniques can be used, for example, to analyse a large number of candidate antibodies to find out which antibody is the most stable. This is also important during formulation studies to identify optimal formulation conditions for stability, such as when considering buffering solutions, pH, excipients and ligands. This allows different conditions to be used and the effect on stability of the protein to be analysed at each different condition.

Whilst measuring UV fluorescence of proteins can be a powerful analysis tool, it is not without its problems. Most notably, the presence of UV-absorbing and/or fluorescing contaminants can detrimentally affect the accuracy of the analysis. For example, if the protein is dissolved in a solvent, it is important to use a solvent which does not, itself, fluoresce under the UV light at, or around 275nm. Problems can also occur if the container used to hold the sample during analysis absorbs UV and also fluoresces. This is especially the case if the apparatus used requires the UV light to pass into a sealed container, such as through a cuvette wall or seal material. This is particularly problematic when using polymeric materials (which often fluoresce) and is conventionally solved using more exotic materials such as quartz (which does not). In addition, when samples are heated it is not possible to direct UV light to the sample directly, as the sample, when heated, can evaporate either leaving no sample or a reduced volume of sample with an increased protein concentration and so a sealed container must be used. This can obviously have a detrimental effect on the accuracy of the results and prevents the use of small sample volumes, which is important for high-throughput analysis. Whilst this can be overcome to some extent by the use of higher solvent volumes, this results in alternative problems and would be considered impractical either because of lack of, or cost, of the sample.

It is, therefore, an object of the present invention to seek to alleviate the above identified problems. SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided an apparatus for analysing a sample, the apparatus comprising:-

(a) a light source, and

(b) a detector, wherein the apparatus is configured for directing light along an excitation path from the light source to a sampling point, and wherein the detector is for detecting fluorescence emitted along an emission path from a sample placed at the sampling point to determine a characteristic of the sample, wherein the apparatus comprises a multi-well plate for holding a plurality of said samples, wherein the multi-well plate comprises a material with a low UV fluorescence background.

Preferably, the apparatus is configured for directing light along an excitation path from the light source and focussing said light to the sampling point.

Preferably, the sampling point is a focal point of the excitation path.

Preferably, the position of the sampling point and/or the multi-well plate is adjustable so that the position of the sampling point corresponds to a well of the or a multi-well plate.

Preferably, the position of the sampling point and/or the multi-well plate is adjustable so that the position of the sampling point corresponds to the centre of the or a well of the or a multi-well plate.

Preferably, the position of the sampling point and/or the multi-well plate is adjustable so that the sampling point is positioned on a central axis of the or a well of the or a multi-well plate.

Preferably, the position of the sampling point and/or the multi-well plate is adjustable so that the position of the sampling point corresponds to the three- dimensional centre of a defined sample volume within the or a well of the or a multi-well plate.

Preferably, the position of the sampling point and/or the multi-well plate is adjustable so that the position of the sampling point can be moved to correspond to a different well of the or a multi-well plate.

Preferably, the position of the sampling point and/or the multi-well plate is adjustable so that the position of the sampling point can be moved from a first well of the or a multi-well plate to a second well of the or a multi-well plate.

Preferably, the position of the sampling point and/or the multi-well plate is adjustable so that the position of the sampling point can be moved to each of a plurality of wells of the or a multi-well plate.

Preferably, the multi-well plate is a 4-well, 6-well, 12-well, 24-well, 48-well, 96- well, 384-well or 1536-well plate.

Preferably, the multi-well plate comprises a polyolefin or a fluoropolymer with a low UV fluorescence background, preferably polypropylene, polyethylene or a fluoropolymer with a low UV fluorescence background.

Preferably, the multi-well plate comprises polypropylene or a fluoropolymer with a low UV fluorescence background.

Preferably, the multi-well plate comprises a transparent fluoropolymer.

Preferably, the multi-well plate comprises ethylene tetrafluoroethylene (ETFE), perfluoroalkoxy alkanes (PFA), ethylene chlorotrifluoroethylene (ECTFE), polyvinylidene difluoride (PVDF), fluorinated ethylene propylene (FEP) or polytetrafluoroethylene (PTFE). Preferably, the multi-well plate comprises fluorinated ethylene propylene (FEP) or polytetrafluoroethylene (PTFE).

Preferably, the multi-well plate comprises a cover.

Preferably, the cover is for sealing one or more, preferably all, of the wells of the multi-well plate.

Preferably, the cover comprises a material with a low UV absorbance and a low UV fluorescence background.

Preferably, the cover comprises a material with a low UV fluorescence background.

Preferably, the cover comprises a polyolefin or a fluoropolymer with a low UV absorbance and low UV fluorescence background, preferably polypropylene, polyethylene or a fluoropolymer with a low UV absorbance and low UV fluorescence background.

Preferably, the cover comprises polypropylene or a fluoropolymer with a low UV fluorescence background.

Preferably, the cover comprises a transparent fluoropolymer.

Preferably, the cover comprises ethylene tetrafluoroethylene (ETFE), perfluoroalkoxy alkanes (PFA), ethylene chlorotrifluoroethylene (ECTFE), polyvinylidene difluoride (PVDF), fluorinated ethylene propylene (FEP) or polytetrafluoroethylene (PTFE).

Preferably, the cover comprises fluorinated ethylene propylene (FEP) or polytetrafluoroethylene (PTFE).

Preferably, the cover is a film, preferably a polymeric film. Preferably, the cover is a film with a thickness in the range of about 50 to about 200 pm, preferably in the range of about 50 to about 100 pm, preferably about in the range of about 50 to about 75pm. Such thicknesses may be used to reduce the UV absorbance and fluorescence of the cover, compared to thicker covers.

Preferably, the cover comprises an adhesive for securing the cover to the multiwell plate.

Preferably, the adhesive is selected from a water-based adhesive, a solvent-based adhesive, a heat-activated adhesive or a pressure-sensitive adhesive.

Preferably, the adhesive is provided on a lower surface of the cover.

Preferably, the adhesive is only applied to the surface of the cover in those areas which will make contact with the multi-well plate.

Preferably, the adhesive comprises a layer of adhesive with a plurality of apertures formed therein.

Preferably, the adhesive comprises a plurality of apertures for allowing (i) a beam of light travelling along the excitation path to pass therethrough and reach a sample held within a well of the multi-well plate, or (ii) a beam of fluorescence travelling from the sample along the emission path to pass therethrough.

Preferably, each aperture covers a surface area of the cover which is at least the size of a well in the multi-well plate and corresponds to the position of a well within the multi-well plate.

Preferably, the apertures correspond to the size and position of wells of the multiwell plate.

Preferably, the apertures do not contain adhesive. Preferably, the light source comprises an LED or a laser.

Preferably, the light source is a UV light source.

Preferably, the light source emits light at a wavelength of between about 250nm and about 300nm, preferably about 275nm.

Preferably, the apparatus is for detecting protein fluorescence.

Preferably, the apparatus is for detecting intrinsic protein fluorescence.

Preferably, the apparatus comprises one or more elements for directing light along the excitation path.

Preferably, the apparatus comprises one or more elements for directing light along the emission path.

Preferably, the apparatus comprises one or more lenses and/or mirrors and/or filters for directing light along the excitation path.

Preferably, the apparatus comprises one or more lenses and/or mirrors and/or filters for directing light along the emission path.

Preferably, the apparatus comprises a collimating lens for directing the light along the excitation path.

Preferably, the apparatus comprises an excitation filter through which light emitted from the light source passes as it travels along the excitation path.

Preferably, the excitation filter allows light to pass through at a wavelength of between about 250nm and about 300nm, preferably about 275nm.

Preferably, the apparatus comprises an excitation beam splitter for splitting light as it travels along the excitation path. Preferably, the excitation beam splitter comprises a dichroic mirror.

Preferably, light having a wavelength greater than an excitation threshold wavelength passes through the excitation beam splitter and light having a wavelength less than the excitation threshold wavelength is reflected by the excitation beam splitter; preferably, wherein the light is reflected in a direction toward the sampling point.

Preferably, the excitation threshold wavelength is at least about 300nm.

Preferably, the excitation beam splitter changes the direction of the excitation path by about 90 degrees. As such, only light having a wavelength less than the excitation threshold wavelength continues along the excitation path.

Preferably, the apparatus comprises an excitation focussing lens for focussing light travelling along the excitation path to the sampling point.

Preferably, light is focussed to the sampling point from above the sampling point. As such, with the provision of a multi-well plate, light travels to the sample from above the multi-well plate.

Preferably, the excitation path comprises a vertical excitation path before the light is focussed to the sampling point.

Preferably, the apparatus is configured for directing fluorescence emitted from a sample along the emission path to the detector.

Preferably, fluorescence which travels along the emission path passes through the excitation focussing lens in an opposite direction to the excitation path.

Preferably, the excitation focussing lens directs fluorescence from the sample along the emission path. Preferably, the excitation focussing lens at least partially collimates fluorescence from the sample.

Preferably, the excitation focussing lens collimates fluorescence from the sample along the emission path.

Preferably, the excitation beam splitter or an emission beam splitter is positioned across the emission path.

Preferably, fluorescence which travels along the emission path passes through the excitation beam splitter or the emission beam splitter.

Preferably, fluorescence having a wavelength greater than the excitation threshold wavelength passes through the excitation beam splitter as it travels along the emission path.

Preferably, fluorescence having a wavelength greater than an emission threshold wavelength passes through the emission beam splitter as it travels along the emission path.

Preferably, the emission threshold wavelength is at least about 300nm.

Preferably, the apparatus comprises an emission focussing lens for focussing fluorescence travelling along the emission path toward the detector.

Preferably, the emission focussing lens is for focussing fluorescence travelling along the emission path to a slit, aperture or fibre-optic input of the detector.

This is particularly advantageous, because it means that a much larger proportion of the fluorescence detected by the detector is from the sample. Preferably, the apparatus comprises a first emission path lens and a second emission path lens, wherein

(i) the first emission path lens is for directing fluorescence from the sample along the emission path towards the second emission path lens, and

(ii) the second emission path lens is for directing fluorescence to a slit, aperture or fibre-optic input of the detector.

Preferably, the first emission path lens is the excitation focussing lens.

Preferably, the second emission path lens is the emission focussing lens.

Preferably, the detector comprises a slit, aperture or fibre-optic input.

Preferably, the slit, aperture or fibre-optic input is between about 12.5pm and about 125pm in width.

Preferably, the slit, aperture or fibre-optic input is between about 12.5pm and about 250pm in length.

As will be appreciated, width refers to the lesser of two dimensions of the slit, aperture or fibre-optic input.

As will be appreciated, length refers to the greater of two dimensions of the slit, aperture or fibre-optic input.

Preferably, the slit, aperture or fibre optic input is rectangular or circular-shaped.

Preferably, the slit, aperture or fibre optic input is rectangular in shape.

Preferably, the slit, aperture or fibre optic input has a width of about 125pm and a length of about 250pm.

Preferably, fluorescence travels along the emission path in a vertical direction away from the sampling point. Preferably, the emission path is a vertical emission path.

Preferably, at least a part of the emission path and at least a part of the excitation path are parallel to each other.

Preferably, light travels along the excitation path as a beam of light.

Preferably, fluorescence travels along the emission path as a beam of fluorescence.

Preferably, the detector comprises a spectrometer.

Preferably, the detector comprises two or more photodetectors.

Preferably, the detector comprises a plurality of photodetectors for producing results across a spectrum of wavelengths.

Preferably, the detector comprises two or more photodiodes.

Preferably, the detector comprises two or more photomultiplier tubes.

Preferably, the detector comprises one or more photodiodes and one or more photomultiplier tubes.

Preferably, the detector and/or the apparatus comprises one or more filters for selecting a range of wavelengths to be detected by the detector.

Preferably, the detector and/or the apparatus comprises a wavelength bandpass optical filter.

Preferably, the detector and/or the apparatus comprises (i) one or more photodiodes and/or one or more photomultiplier tubes, and (ii) a filter for selecting a range of wavelengths, wherein the filter is positioned in front of the one or more photodiodes and/or one or more photomultiplier tubes.

Preferably, the filter is a wavelength bandpass optical filter.

Preferably, the selected range of wavelengths is between about 310 nm and about 435 nm.

Preferably, the detector is for detecting fluorescence.

Preferably, the detector is for detecting fluorescence at wavelengths between about 310 nm and about 435 nm.

Preferably, the detector collects fluorescent signal at discrete wavelengths.

Preferably, the discrete wavelengths are from below and above the wavelength of peak intensity such that the ratio of signals from each wavelength is indicative of a shift in the wavelength of peak intensity.

Preferably, the discrete wavelengths are one below and one above the wavelength of peak intensity such that the ratio of signals from each wavelength is indicative of a shift in the wavelength of peak intensity.

Preferably, the apparatus comprises a multi-well plate support for holding the or a multi-well plate.

Preferably, the support comprises a platform.

Preferably, the support comprises a support plate or tray.

Preferably, the support is for holding a 4-well, 6-well, 12-well, 24-well, 48-well, 96-well, 384-well and/or 1536-well plate. Preferably, the support is moveable to position the sampling point within a well of the or a multi-well plate held by the support.

Preferably, the support is moveable to alter the position of the sampling point, preferably in an x-y plane.

Preferably, the support is moveable to locate the or a multi-well plate in a plurality of different positions, wherein the sampling point is located in a different well of the multi-well plate in each position.

Preferably, the excitation path is moveable to position the sampling point within a well of the or a multi-well plate.

Preferably, the excitation path is moveable to locate the or a multi-well plate in a plurality of different positions, wherein the sampling point is located in a different well of the multi-well plate in each position.

Preferably, one or more components of the apparatus are moveable to alter the position of the sampling point, preferably in an x-y plane.

Preferably, one or more of the light source, the detector, the support, the collimating lens, the excitation filter, the excitation beam splitter, the excitation focussing lens, the emission beam splitter and/or the emission focussing lens are provided on a moveable component support.

Preferably, reference to "one or more of the components of the apparatus" means one or more of the light source, the detector, the support, the collimating lens, the excitation filter, the excitation beam splitter, the excitation focussing lens, the emission beam splitter and/or the emission focussing lens.

Preferably, the apparatus comprises a temperature controlling device for controlling the temperature of the or a multi-well plate. As such, the temperature controlling device controls the temperature of one or more samples each held within a well of the or a multi-well plate. Preferably, the temperature controlling device is for controlling the temperature of the or a multi-well plate in the range of about 4 °C to about 120 °C, preferably in the range of about 20 °C to about 100 °C, preferably in the range of about 40 °C to about 90 °C. Preferably, the temperature control device can be used to set a pre-defined temperature within the range. Preferably, the temperature controlling device can change the temperature of the device at a defined rate.

Preferably, the temperature controlling device is for abutment with the or a multiwell plate.

Preferably, the temperature controlling device is for engagement with the or a multi-well plate or for positioning adjacent the or a multi-well plate, wherein the temperature of the temperature controlling device is altered to control the temperature of the multi-well plate and/or one or more samples held therein.

Preferably, the temperature controlling device comprises one or more surfaces for engagement with the or a multi-well plate or for positioning adjacent the or a multi-well plate, wherein the temperature of the one or more surfaces is controlled to control the temperature of the multi-well plate and/or one or more samples held therein.

Preferably, the temperature of the temperature controlling device, or the one or more surfaces thereof, is set at a greater temperature than the desired temperature of the or a multi-well plate.

Preferably, the temperature controlling device, or the one or more surfaces thereof, is shaped to correspond to or mate with the shape of the or a multi-well plate.

Preferably, the temperature controlling device, or the one or more surfaces thereof, comprises a plurality of apertures corresponding to the size and position of wells in the or a multi-well plate. Preferably, the temperature controlling device comprises a base.

Preferably, the base is for heating a multi-well plate from below.

Preferably, the base comprises a plate or block.

Preferably, a surface of the temperature controlling device is provided on the base.

Preferably, the temperature controlling device comprises a thermoelectric heat pump. This has an advantage of heating and cooling the multi-well plate.

Preferably, the apparatus comprises a lid for the or a multi-well plate.

Preferably, the lid is heated.

Preferably, the lid forms part of the temperature controlling device.

Preferably, a surface of the temperature controlling device is provided on the lid.

Preferably, the lid is for heating the or a multi-well plate from above.

Preferably, the temperature controlling device comprises a base for heating the or a multi-well plate from below and a lid for heating the or a multi-well plate from above.

Preferably, the lid for heating the or a multi-well plate comprises a heater.

Preferably, in use, the lid is heated to a higher temperature than the base. This reduces condensation or prevents condensation forming on the surface of a cover positioned over the or a multi-well plate. Preferably, the lid for heating is heated to a temperature in the range of about 100 °C to about 120°C.

Preferably, the lid controls the temperature of a sample within a well of the or a multi-well plate.

Preferably, the lid comprises a material with a low UV fluorescence background.

Preferably, the lid comprises a thermally conductive material.

Preferably, the lid comprises a metal.

Preferably, the lid comprises aluminium or stainless steel.

Preferably, the lid comprises a plurality of apertures for allowing (i) a beam of light travelling along the excitation path to pass therethrough and reach a sample held within a well of the or a multi-well plate, or (ii) a beam of fluorescence travelling from the sample along the emission path to pass therethrough.

Preferably, each aperture covers a surface area of the lid which is at least the size of a well in the or a multi-well plate and corresponds to the position of a well within the or a multi-well plate.

Preferably, the apertures correspond to the size and position of wells of the or a multi-well plate.

Preferably, the lid is positioned over the cover.

Preferably, the cover is positioned between the multi-well plate and the lid.

Preferably the lid for heating heats the cover. Surprisingly, heating the cover results in a reduction of UV fluorescence by the cover. This is shown in the examples. According to another aspect of the present invention, there is provided an apparatus for analysing a sample, the apparatus comprising:-

(a) a light source, and

(b) a detector, wherein the apparatus is configured for directing light along an excitation path from the light source and focussing said light to a sampling point, and wherein the detector is for detecting fluorescence emitted along an emission path from a sample placed at the sampling point to determine a characteristic of the sample, wherein the apparatus is configured for restricting the width of the excitation path as the light is focussed to the sampling point such that when the sampling point is within the well of a multi-well plate, the excitation path does not pass through a wall of the multi-well plate before it reaches the sample.

Preferably, the apparatus comprises a lens for restricting the width of the excitation path as the light is focussed to the sampling point such that when the sampling point is within the well of a multi-well plate, the excitation path does not pass through a wall of the multi-well plate before it reaches the sample.

Preferably, the position of the sampling point and/or a multi-well plate used with the apparatus is adjustable so that the position of the sampling point corresponds to a well of the or a multi-well plate.

Preferably, the position of the sampling point and/or a multi-well plate used with the apparatus is adjustable so that the position of the sampling point corresponds to the centre of the or a well of the or a multi-well plate.

Preferably, the position of the sampling point and/or a multi-well plate used with the apparatus is adjustable so that the sampling point is positioned on a central axis of the or a well of the or a multi-well plate. Preferably, the position of the sampling point and/or a multi-well plate used with the apparatus is adjustable so that the position of the sampling point corresponds to the three-dimensional centre of a defined sample volume within the or a well of the or a multi-well plate.

Preferably, the position of the sampling point and/or a multi-well plate used with the apparatus is adjustable so that the position of the sampling point can be moved to correspond to a different well of the or a multi-well plate.

Preferably, the position of the sampling point and/or a multi-well plate used with the apparatus is adjustable so that the position of the sampling point can be moved from a first well of the or a multi-well plate to a second well of the or a multi-well plate.

Preferably, the position of the sampling point and/or a multi-well plate used with the apparatus is adjustable so that the position of the sampling point can be moved to each of a plurality of wells of the or a multi-well plate.

Preferably, the apparatus comprises a multi-well plate.

Preferably, the multi-well plate is a multi-well plate as described herein.

Preferably, the multi-well plate comprises a material with a low UV fluorescence background.

According to another aspect of the present invention, there is provided a method for analysing a plurality of samples, the method comprising:-

(i) providing a plurality of samples wherein each sample is positioned in a well of a multi-well plate,

(ii) analysing the samples using an apparatus which comprises

(a) a light source, and

(b) a detector, wherein the apparatus is configured for directing light along an excitation path from the light source to a sampling point, and wherein the detector is for detecting fluorescence emitted along an emission path from a sample placed at the sampling point to determine a characteristic of the sample, wherein the multi-well plate comprises a material with a low UV fluorescence background.

Preferably, the apparatus is an apparatus as described herein.

According to another aspect of the present invention, there is provided a method for analysing a plurality of samples, the method comprising:-

(i) providing a plurality of samples in wells of a multi-well plate, and

(ii) analysing the samples using an apparatus as described herein.

Preferably, the multi-well plate is a multi-well plate as described herein.

According to another aspect of the present invention, there is provided the use of a heated lid to reduce the UV fluorescence of a cover, wherein the cover is for a multi-well plate for holding a plurality of samples, wherein the multi-well plate comprises a material with a low UV fluorescence background.

Surprisingly, heating the cover results in a reduction of UV fluorescence by the cover. This is shown in the examples.

Preferably, wherein the heated lid heats the cover to a temperature in the range of about 100 °C to about 120 °C.

Preferably, the cover comprises a material with a low UV absorbance and a low UV fluorescence.

Preferably, the cover comprises a polyolefin or a fluoropolymer with a low UV fluorescence background, preferably polypropylene, polyethylene or a fluoropolymer with a low UV fluorescence background, preferably polypropylene or a fluoropolymer with a low UV fluorescence.

Preferably, the multi-well plate is a multi-well plate as described herein. Preferably, the cover is a cover as described herein.

Within this specification embodiments have been described in a way which enables a clear and concise specification to be written, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the invention. For example, it will be appreciated that all preferred features described herein are applicable to all aspects of the invention described herein and vice versa.

Within this specification, the term "about" means plus or minus 20%, more preferably plus or minus 10%, even more preferably plus or minus 5%, most preferably plus or minus 2%.

It will be appreciated that reference to "one or more" includes reference to "a plurality".

Within this specification, reference to "a low UV fluorescence background" means that the material by itself, when excited by 275nm has a maximum of three times the water raman peak recorded at 320nm.

Preferably, reference to "a material with a low UV fluorescence background" means a polyolefin or a fluoropolymer with a low UV fluorescence background, preferably polypropylene, polyethylene or a fluoropolymer with a low UV fluorescence background.

Preferably, reference to "a material with a low UV fluorescence background" means polypropylene or a fluoropolymer with a low UV fluorescence background.

Preferably, reference to "a material with a low UV fluorescence background" means a transparent fluoropolymer.

Preferably, reference to "a material with a low UV fluorescence background" means ethylene tetrafluoroethylene (ETFE), perfluoroalkoxy alkanes (PFA), ethylene chlorotrifluoroethylene (ECTFE), polyvinylidene difluoride (PVDF), fluorinated ethylene propylene (FEP) or polytetrafluoroethylene (PTFE).

Preferably, reference to "a material with a low UV fluorescence background" means fluorinated ethylene propylene (FEP) or polytetrafluoroethylene (PTFE).

Preferably, reference to "a material with a low UV fluorescence background" means a polymer material which does not comprise a plasticiser, preferably wherein the plasticiser comprises an aromatic group.

Within this specification, reference to "low UV absorbance" means less than 0.05 absorbance at the wavelength range 270-310nm.

Preferably, reference to "a material with a low UV absorbance" means a polyolefin or a fluoropolymer with a low UV fluorescence background, preferably polypropylene, polyethylene or a fluoropolymer with a low UV fluorescence background.

Preferably, reference to "a material with a low UV absorbance" means polypropylene or a fluoropolymer with a low UV fluorescence background.

Preferably, reference to "a material with a low UV fluorescence background" means polypropylene or a fluoropolymer with a low UV fluorescence background.

Preferably, reference to "a material with a low UV absorbance" means a transparent fluoropolymer.

Preferably, reference to "a material with a low UV absorbance" means ethylene tetrafluoroethylene (ETFE), perfluoroalkoxy alkanes (PFA), ethylene chlorotrifluoroethylene (ECTFE), polyvinylidene difluoride (PVDF), fluorinated ethylene propylene (FEP) or polytetrafluoroethylene (PTFE).

Preferably, reference to "a material with a low UV absorbance" means fluorinated ethylene propylene (FEP) or polytetrafluoroethylene (PTFE). Preferably, reference to "a material with a low UV absorbance" means a polymer material which does not comprise a plasticiser, preferably wherein the plasticiser comprises an aromatic group.

Preferably, the apparatuses and methods described herein are for detecting fluorescence in a sample.

Preferably, the apparatuses and methods described herein are for detecting protein fluorescence in a sample.

Preferably, the apparatuses and methods described herein are for detecting intrinsic protein fluorescence in a sample.

DETAILED DESCRIPTION

Example embodiments of the present invention will now be described with reference to the accompanying Figures, in which

Figure 1 shows a schematic arrangement of an apparatus according to the present invention;

Figure 2 shows the excitation path (Figure 2A) and emission path (Figure 2B) of a schematic arrangement of an apparatus according to the present invention;

Figure 3A shows a multi-well plate for use in the present invention;

Figure 3B shows a view from below the cover of the multi-well plate shown in Figure 3A; and

Figure 4 shows an exploded view of multi-well plate and a temperature controlling device.

Figure 5 shows the UV fluorescence signal to background with temperature of the cover of a multi-well plate.

Figure 6 shows the UV fluorescence background signal of a cover of a multi-well plate with temperature. The present invention relates to an apparatus for analysing proteins in a sample, in particular for detecting protein fluorescence, in particular intrinsic protein fluorescence.

The apparatus relies upon the use of UV light to excite electrons in the protein molecules, which then causes them to emit fluorescence.

With reference to Figure 1, a schematic drawing showing an example layout of an apparatus 1 of the present invention is provided.

Light is emitted from a 275nm UV LED light source 2 along an excitation path, Ex, and passes through an LED collimating lens 3. The collimated light then passes through an excitation filter 4 and is then reflected by a dichroic mirror 5 toward an objective lens 6. The excitation filter 4 filters out light which is not at 275nm and so only light having a wavelength of 275nm passes through and further along the excitation path. In the example shown, the dichroic mirror 5 is a UV reflective beam splitter with a threshold above 300nm. The objective lens 6 focusses the light to a point corresponding to the centre of a sample in the well of a multi-well plate 7. The ability of the apparatus to focus the UV light is particularly advantageous because it means that UV light from the LED source is only directed to the sample being analysed. This greatly reduces the amount of background emission from both the multi-well plate 7 itself and any other possible contaminants associated with the apparatus.

The UV light is absorbed by tryptophan and tyrosine residues of the protein present within the sample in a well of the micro-well plate 7, which results in the emission of fluorescence along an emission path, EM.

Fluorescence from the sample passes along the emission path Em and through the objective lens 6, which acts as a collimating lens for the fluorescence passing along the emission path. Collimated fluorescence having a wavelength greater than the threshold of the dichroic mirror 5 passes through the dichroic mirror 5 and then through an emission focussing lens 8, which focusses the fluorescence for detection by the detector 9. With reference to Figure 2B, the fluorescence is focussed onto a slit/aperture 23 of the detector 9, however, it will be appreciated that it could also or alternatively be focussed to a fibre-optic input of the detector 9.

In the example shown, the detector is a spectrometer which detects fluorescence at wavelengths between 310 nm and 435 nm.

Focussing of the fluorescence from the emission path onto a slit, aperture or fibreoptic input of the detector greatly reduces the amount of general background fluorescence analysed by the detector. In this respect, the vast majority of the fluorescence which passes through the slit, aperture or fibre-optic input of the detector will be from the sample.

It will be appreciated that multi-well plates of various sizes could be used, including 4-well, 6-well, 12-well, 24-well, 48-well, 96-well, 384-well or even 1536- well plates.

Alternative views of the arrangement of the apparatus 1 are shown in Figures 2A and 2B in which Figure 2A shows the excitation path Ex in greater detail and Figure 2B shows the emission path Em in greater detail. In Figures 2A and 2B a sample 10 is shown within a well 11 of the multi-well plate 7.

As will be noted from the arrangement shown in Figures 1, 2A and 2B, fluorescence emitted from the sample passes along the emission path Em through the same lens 6 through which the excitation path Ex passes. This means that the sample 10 is contacted from above by light from the LED source and then fluorescence emitted by the sample passes along the emission path Em in the opposing direction. As a result, neither the excitation path nor the emission path pass through the walls of the multi-well plate 7. This is advantageous because it reduces fluorescence contamination, which could occur if the walls of the multiwell plate 7 were caused to emit fluorescence following excitation thereof by light from the light source 2. As will be appreciated, by avoiding this source of contamination, the accuracy of the results obtained with the apparatus is greatly improved.

Whilst multi-well plates are usually manufactured from polystyrene, this fluoresces when excited by UV light. As a result, the use of standard multi-well plates can result in background fluorescence. Whilst this is greatly improved by the arrangement described above with vertically orientated excitation and emission paths and a focussed excitation path, this can be improved further by the use of multi-well plates manufactured from a material having a low UV fluorescence background.

The most effective materials for this purpose are a polyolefin, or a fluoropolymer with a low UV fluorescence background, for example polypropylene, polyolefin, polyethylene or a fluoropolymer with a low UV fluorescence background, for example polypropylene or a fluoropolymer with a low UV fluorescence background. For example, whilst a fluorinated ethylene propylene (FEP) multi-well plate was used in the example shown, ethylene tetrafluoroethylene (ETFE), perfluoroalkoxy alkanes (PFA), ethylene chlorotrifluoroethylene (ECTFE), polyvinylidene difluoride (PVDF), or polytetrafluoroethylene (PTFE) could also be used as the material for the multi-well plate.

In order to prevent the sample evaporating from the multi-well plate, a seal can be provided. This is shown in Figure 3A in which a 384-well plate 7 has been provided with a cover 12. In the example shown, both the multi-well plate 7 and the cover 12 have been manufactured from FEP.

The cover 12 includes an adhesive layer 13 provided on its lower surface, that is the surface facing the multi-well plate 7. However, in order to prevent the emission and excitation paths being impeded by the presence of the adhesive 13, which would also cause fluorescence contamination, the adhesive 13 is only applied to the surface of the cover 12 in those areas which will make contact with the upper surface of the multi-well plate 7. As a result, the adhesive layer 13 comprises a plurality of apertures 14 which correspond to the position of the wells 11 of the multi-well plate 7. As a result, the use of the adhesive 13 to secure the cover 12 to the multi-well plate 7 does not interfere with the emission and excitation paths and does not result in fluorescence contamination.

In order to minimise fluorescence contamination, the material used for the cover should be carefully chosen. A cover comprising FEP was used in the example shown, however, as with the multi-well plate, polypropylene or a fluoropolymer with a low UV fluorescence background, for example ETFE, PFA, ECTFE, PVDF, or PTFE, could also be used. Further, the cover could comprise a polyolefin or a fluoropolymer with a low UV fluorescence background, such as polyethylene, polypropylene or a fluoropolymer with a low UV fluorescence background.

In order to control the temperature of a multi-well plate and thus of samples held within the multi-well plate, the apparatus may include a temperature controlling device 15 for controlling the temperature of the multi-well plate 7.

Figure 4 shows a multi-well plate 7 with a cover 12 comprising an adhesive layer 13 for securing the cover 12 to the multi-well plate. In order to control the temperature of the multi-well plate 7, and thus samples held within the multi-well plate 7, a temperature controlling device 15 is provided.

The temperature controlling device 15 includes a first heating surface 16 provided on a block 17 which engages with the underside of the multi-well plate 7 and which is shaped to match the shape of the multi-well plate 7. In particular, the block 17 includes a plurality of recesses 18 for accommodating the wells 11 of the multiwell plate 7.

The temperature controlling device 15 also includes a lid 20 which includes a second heating surface 22 for heating the multi-well plate 7 from above. The lid 20 is heated to a higher temperature than the block 17 which helps to prevent the build-up of condensation on the inside surface of the cover 12. In practice, it is the lid 20 which controls the temperature of a sample held within a well 11 of the multi-well plate 7. The lid can contain a printed circuit board or a thin film heater to heat the second heating surface 22. The printed circuit board or thin film heater could, for example, be provided on top of the second heating surface 22.

The temperature controlling device 15 also includes a thermoelectric heat pump 19.

The lid 20 includes a plurality of apertures 21 formed therein to allow the unimpeded passage of light to a sample held within a well of the multi-well plate and for the unimpeded passage of fluorescence from the sample along the emission path. In the example shown, the lid 20 is an aluminium plate, for example with a thin film heater, although it could also be manufactured from other materials, such as stainless steel.

Whilst not specifically shown in the Figures, it will be appreciated that the apparatus 1 may include a support for holding the multi-well plate. The support could, for example, take the form of a platform, plate or tray suitable for holding a multi-well plate, for example a 4-well, 6-well, 12-well, 24-well, 48-well, 96- well, 384-well and/or 1536-well plate.

The support could take the form of a platform for holding the multi-well plate with the platform moveable in the x and y directions to move the multi-well plate. It is preferred that the multi-well plate is moved in the x, / direction while maintaining the same vertical position. The support is moveable to locate the multi-well plate in a plurality of different positions, wherein the sampling point is located in a different well of the multi-well plate in each position.

It will also be appreciated that, instead of moving the multi-well plate, the apparatus could be configured to allow the position of the sampling point to be moved between different wells of the multi-well plate. This could, for example, be achieved by allowing movement of one or more components of the apparatus, for example one or more of the optical elements. In one example, the optical elements could be provided on a support, with the support moveable to move the position of the sampling point. In examples of the present invention which include a temperature controlling device 15, a support or platform could be provided in a form which moves the stack of temperature controlling device 15 with lid 20 and multi-well plate 7 with cover 12, as shown in Figure 4.

EXAMPLES

Example 1

A multi-well microplate was filled with two different samples. Ten wells were filled with phosphate buffered saline (PBS). Forty wells were filled with tryptophan. The multi-well plate was sealed with a cover of polyolefin film with a pressure sensitive adhesive. The multi-well microplate was heated to the following temperatures, 25 °C, 30 °C, 40°C , 50 °C, 60 °C, 70 °C, 80 °C, 90 °C, 100 °C, 110 °C and 115 °C. The UV fluorescence (excitation 275nm, emission at 330nm) of the microplate was scanned at each temperature as the microplate was heated to 115 °C and again at each temperature as the microplate was cooled back to 25 °C.

Figure 5 shows that the signal to background increases as the temperature of the cover increases. This shows that the background signal, that is the UV fluorescence of the well and the cover, decreases as the temperature of the cover increases. Figure 5 also shows the same pattern as the cover is cooled, with the background signal increasing as the cover is cooled. It is a surprising advantage of the invention that the UV fluorescence of the cover decreases as the temperature of the cover increases.

Figure 6 shows that the background fluorescence signal of the cover is reduced from 1,741 to 654 counts when the cover is heated from 25 °C to 115 °C.

It should be understood that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications can be made without departing from the spirit and scope of the present invention and without diminishing its attendant advantages. It is therefore intended that such changes and modifications are covered by the appended claims.