WO2007126389A1

WO2007126389A1 - Optical detector system for sample analysis having at least two different optical pathlengths

Info

Publication number: WO2007126389A1
Application number: PCT/SG2006/000114
Authority: WO
Inventors: Chu-Sing Lim; Aneira Marissa Pariwono; Pak-Chiang Yip
Original assignee: Asensor Pte Ltd
Priority date: 2006-05-02
Filing date: 2006-05-02
Publication date: 2007-11-08

Abstract

The invention relates to a cuvette and a cuvette holder. The cuvette (5) includes a sample compartment (5) with an inlet (4). The sample compartment is defined by a circumferential wall (3) and a base (2). The circumferential wall has a light incident wall portion and a light emerging wall portion. The light emerging wall portion is opposite to the light incident wall portion. The sample compartment (5) is designed such that it has at least two different optical pathlengths between the light incident wall portion and the light emerging wall portion. The cuvette holder (Figs. 5A-5D) is designed to match the outer shape of said cuvette.

Description

OPTICAL DETECTOR SYSTEM FOR SAMPLE ANALYSIS HAVING AT LEAST TWO DIFFERENT OPTICAL PATHLENGTHS

[0001] The present invention relates to an optical detector system for sample analysis, in particular a cuvette, that has at least two different optical pathlengths.

[0002] Optical detection often includes measurements of a sample of interest in a spectrophotometer. A light beam of a selected wavelength is transmitted from a light source to a respective sample where the radiation is absorbed. Upon irradiation the sample may itself emit light. Different substances absorb or emit light in different ways. The intensity of light absorbed or emitted by the sample is thus measured at a detector. This may include recording how the intensity changes as a function of the energy, wavelength, or frequency of the light.

[0003] The sample, generally a fluid or included in a fluid, is typically provided in a cuvette (French: "basin"). A cuvette is a container, usually a small square tube made of plastic, glass, or quartz that is sealed at one end. A cuvette has at least two opposing walls that permit light to pass through, typically of as high transparency as possible. While for fast measurements often disposable inexpensive plastic cuvettes are used, accurate measurements require the use of highly pure glass or quartz material, with as few impurities as possible, as these may affect a spectroscopic reading. [0004] The sample chamber of a cuvette has a standard optical pathlength, i.e. distance between the points where the light enters and leaves the medium of a respective sample, of 10 mm and typical cuvettes are of an outside dimension of 12 mm x 12 mm. Many cuvettes, in particular if they are to be used in fluorescence spectroscopy, are transparent on all four sides (cf. e.g. Fig. 3A). Other cuvettes are transparent only on two opposite sides, so that they allow a beam of light to pass only through the respective pair of sides (cf. e.g. Fig. 3B). The opaque or non-transparent sides of such cuvettes often have ridges or are rough to allow easy handling. Two examples of a cuvette with only two transparent opposite sides are a semi-micro cuvette (cf. Figures 3C and 3D) and a micro cuvette (cf . Fig. 3E).

[0005] Matter that absorbs light in the spectral region of electromagnetic radiation of interest is known as a chromophore. The absorption of light intensity in an ideal non-scattering sample and a non-absorbing medium can be described by the Lambert-Beer Law. According to this law the attenuation (A) caused by an absorbing compound is proportional to the concentration of the compound in the solution (c) and the optical pathlength (d): A = loglO [lo/I] = ε • c • d wherein A is the attenuation measured in optical densities, Io and I are the light intensities incident on and emerging from the medium, respectively, ε is the specific extinction coefficient of the absorbing compound, c is the concentration of the absorbing compound in the solution, and d is the optical pathlength.

[0006] For non-ideal samples typically scattering occurs, which is a change in the direction of the light while passing through the sample. This is usually due to sample inhomogeneity, which may for example be described as a plurality of variations in refractive index within the sample. As a result the pathlength travelled by photons within the sample can be substantially increased, which is equivalent to a significantly increase in the probability of absorption occurring. Due to the deflection, scattering furthermore results in a decreased signal to noise ratio during detection of the light that emerges from the sample. Overall scattering leads to undesired errors in measurement. Since sample inhomogeneity generally varies from sample to sample, these errors likewise vary between samples, thus complicating or instance a comparison between samples of different origin.

[0007] Attempts to overcome the problem of scattering have so far focused on the use of focusing lenses, absorption filters or multiple light beams entering the cuvette from different directions. The use of absorption filters is disclosed in International Patent Application WO 01/53806. The use of multiple light beams and detection optics in conjunction with cross- correlation technique is disclosed in US patent applications 2003/0128363, 2005/0185176, and 2005/0185177 for example. However, the use of additional means, such as light beams of filters, bears the risk of additional undesired errors in measurement and furthermore complicates measurements. There thus remains a need for a simple means of improving optical detection accuracy, in particular of samples of varying scattering properties. [0008] Accordingly, it is an objective of the present invention to provide a device that addresses the above described problems without the requirement of introducing further filters or light beams.

[0009] This objective is solved by a cuvette according to claim 1.

[0010] In one aspect the present invention provides a cuvette. The cuvette includes a sample compartment with an inlet. The sample compartment is defined by a circumferential wall and a base. The circumferential wall has a light incident wall portion and a light emerging wall portion. The light emerging wall portion is opposite to the light incident wall portion. The sample compartment is designed such that it has at least two different distances between a point where light enters the sample compartment via the light incident wall portion and a point where light emerges from the sample compartment via the light emerging wall portion. Accordingly, the sample compartment has at least two different optical pathlengths between the light incident wall portion and the light emerging wall portion. [0011] In a further aspect the invention provides a cuvette holder. The cuvette holder is capable of accommodating a cuvette as defined above. The cuvette holder is designed to match the outer shape of the cuvette.

[0012] In yet a further aspect the invention provides an apparatus for measuring the absorption and/or emission of light in a fluid sample. The apparatus is capable of accommodating one or at least one cuvette.

[0013] The cuvette of the present invention includes a sample compartment. The sample compartment may be of any capacity. The capacity may for instance be selected in the range of about 0.01 to about 15 ml, such as in the range of about 0.05 to about 10 ml, for example in the range of about 0.1 to about 5 ml or in the range of about 0.05 to about 2 ml. The sample compartment may be designed to be capable of accommodating any desired sample (cf. also below). Generally the sample is a fluid or included in a fluid. The fluid may be of any properties, whether polar or apolar. Typically a sample is a liquid or a gas or included therein. Examples of a respective gas include, but are not limited to air, nitrogen, argon, helium, hydrogen, hydrogen sulphide and carbon dioxide. [0014] Examples of nonpolar aprotic liquids include, but are not limited to, hexane, heptane, cyclohexane, benzene, toluene, pyridine, dichloromethane, chloroform, carbon tetrachloride, carbon disulfide, tetrahydrofuran, dioxane, diethyl ether, diisopropylether, ethylene glycol monobutyl ether or tetrahydrofuran. Examples of dipolar aprotic liquids include, but are not limited to, methyl ethyl ketone, methyl isobutyl ketone, acetone, cyclohexanone, ethyl acetate, isobutyl isobutyrate, ethylene glycol diacetate, dimethylformamide, acetonitrile, N,N-dimethyl acetamide, nitromethane, acetonitrile, N- methylpyrrolidone, and dimethylsulfoxide. Examples of polar protic liquids include, but are not limited to, water, methanol, ethanol, butyl alcohol, formic acid, dimethylarsinic acid [(CHs)₂AsO(OH)], N,N-dimethyl-formamide, N,N-diisopropylethylamine, or chlorophenol. Examples of nonpolar protic liquids include, but are not limited to, acetic acid, tert.-butyl alcohol, phenol, cyclohexanol, or aniline. Two illustrative examples of ionic liquids are 1,3- dialkylimidazolium-tetrafluoroborates and 1,3-dialkylimidazolium-hexafluoroborates.

[0015] The sample compartment of the cuvette has an inlet. Through the inlet a respective sample may be disposed into, or enter, the sample compartment. In some embodiments a respective sample can also be removed from, or leave, the sample compartment via the inlet. The inlet may be located at any desired position relative to a selected point or region of the sample compartment. The inlet may for example be located on a top of the sample compartment. The terms "on top of, "on a top of, "at a bottom of, "at the bottom of, "below" and "above" as used herein refer to a position where the cuvette is placed into e.g. a spectrophotometer, a spectropolarimeter or a fluorometer, i.e. in which a measurement is typically carried out. Generally in such a position the base (cf. below) is located at a bottom of the sample compartment. In on one embodiment the inlet is located on a top of the sample compartment and is at least essentially opposite to the base. The inlet may be of any form and dimension. Examples of an inlet include, but are not limited to, a valve, a chamber, a neck or a channel. In some embodiments the inlet is an opening. A respective opening may be of any shape, profile and diameter. The cuvette may further include a seal for the inlet. In one of the embodiments where the inlet is an opening, the cuvette includes a seal for the respective opening. The seal, which may be removable from the cuvette (cf. e.g. Fig. 2), may for instance prevent matter from entering or leaving the sample compartment. A respective seal may include any desired material. Three purely illustrative examples of materials that may be used are glass, polypropylene (PP) and polytetrafluoroethylene (PFTE, Teflon).

[0016] The sample compartment is defined by a circumferential wall and a base. The circumferential wall and the base may include or consist of any material, as long as the circumferential wall includes a light incident wall portion and a light emerging wall portion (cf . below). It may be desired to select a respective material that is capable of withstanding the conditions of a desired optical measurement. It may also be desired to select a material that does not affect, interfere with or retard a desired optical measurement. As an example, it may be desired to select a material that withstands light of a selected wavelength, for instance UV light. As a further example it may be desired to select a material that maintains its consistency at a selected temperature, or that is inert against a selected sample such as acid or alkali.

Depending on the selected materials, the cuvette may be designed to be disposable or reusable.

[0017] The circumferential wall and the base may be of any geometry and dimension. They may for instance be curved, round, straight or flat. It is understood that the sample compartment of the cuvette is defined by the circumferential wall, which may be of any thickness. Accordingly, the geometry of the outer shape of a cuvette of the invention may differ from the geometry of the sample compartment. As an illustrative example, the outer shape of the cuvette depicted in Fig. IF is the shape of a cuboid with four lateral straight outer walls. One of the lateral outer walls of the cuvette contains inwardly directed ledges. Nevertheless the outer shape of the cuvette depicted in Fig. IF is of uniform square cross section along the height of the cuvette, in a level parallel to the base. The sample compartment of the cuvette depicted in Fig. IF is defined by one circumferential wall, which corresponds to the inner surfaces of the four lateral outer walls of the cuvette. In contrast to the outer shape of the cuvette, the circumferential wall of the sample compartment has a plurality of different cross sections along the height of the sample compartment. In direct vicinity to the base and the inlet, the cross section of the sample compartment is for instance square, while at a level at about equal distance from the base and from the inlet, the cross section of the sample compartment is oblong.

[0018] In some embodiments the circumferential wall of the sample compartment includes at least two wall portions of different geometry and/or orientation. Such wall portions may include a light incident wall portion and/or a light emerging wall portion (cf. also below) or a section thereof. As an illustrative example, a cuvette of the present invention may include a circumferential wall with at least two lateral wall portions that are inclined with respect to each other. At least one of these wall portions may for instance be a straight wall. As already indicated above, both the circumferential wall and the base may be a part of another, typically larger, wall or base, respectively. The circumferential wall and the base may be orientated in any angle with respect to each other, for example in an angle that is larger than 0° and smaller than 180°, such as an angle that is larger than 0° and smaller than about 90°. In typical embodiments (cf. e.g. Fig. 1 and Fig. 2) the circumferential wall is a lateral wall.

[0019] A cuvette is used for optical measurements (cf. above), generally for determining the absorption and/or emission of a sample, which is disposed in the sample compartment. Accordingly, it is usually desired to expose the sample to light from a light source for optical detection, for instance in a spectrophotometer or a fluorometer. The term "light" is understood to include electromagnetic radiation of any wavelength, including a distinct wavelength, a set of distinct wavelengths or any region of the electromagnetic spectrum. Two examples of a region of the electromagnetic spectrum are visible light, corresponding to a wavelength range of about 400 to about 700 nanometers, and ultraviolet light, corresponding to a wavelength range of about 30 to about 400 nanometers. Accordingly the sample compartment of the cuvette of the invention includes at least two wall portions that allow light to enter into and emerge from the sample compartment. One of these two wall portions is a light incident wall portion, which allows light to enter the sample compartment. The other of these two wall portions is a light emerging wall portion, which is opposite to the light incident wall portion and allows light to leave the sample compartment. Hence, a light beam can enter the sample compartment via the light incident wall portion and emerge from the sample compartment via the light emerging wall portion. In some embodiments the light incident wall portion, or a section thereof, may furthermore also be designed to be capable of allowing light to emerge from the sample compartment. In such embodiments the light incident wall portion (or a respective section thereof) may also be used as a light emerging wall portion where desired. Likewise, in some embodiments the light emerging wall portion, or a section thereof, may be designed to be capable of allowing light to enter into the sample compartment. In such embodiments the light emerging wall portion (or a respective section thereof) may also be used as a light incident wall portion where desired.

[0020] As indicated above, the light incident wall portion and the light emerging wall portion allow at least a certain percentage of light to pass through. The light incident wall portion and the light emerging wall portion may independently from each other allow light of a defined wavelength, or light within a certain range of the electromagnetic spectrum, for example visible light, infrared light, X-ray and/or UV light, to pass through.

[0021] In some embodiments the light incident wall portion only allows light of a certain wavelength, or certain wavelengths, to enter the sample compartment. Furthermore, in some embodiments the light incident wall portion also allows, at least to a certain extent, light of a certain wavelength, or certain wavelengths, to emerge from the sample compartment. In some embodiments the wavelength, or wavelengths of light that the light incident wall portion allows to enter the sample compartment, and the wavelength, or wavelengths of light that the light incident wall portion allows to leave the sample compartment are identical. In one embodiment the light incident wall portion allows light of the same wavelength or wavelengths to enter and leave the sample compartment to the same extent. [0022] In some embodiments the above said also applies to the light emerging wall portion, independent of the light incident wall portion. Accordingly, in some embodiments the light emerging wall portion only allows light of a certain wavelength, or certain wavelengths, to emerge from the sample compartment. In some embodiments the light emerging wall portion also allows light of a certain wavelength, or certain wavelengths, to enter the sample compartment, at least to a certain extent. In some embodiments the wavelength, or wavelengths of light that the light emerging wall portion allows to emerge from the sample compartment, and the wavelength, or wavelengths of light that the light emerging wall portion allows to enter the sample compartment are identical. In one embodiment the light emerging wall portion allows light of the same wavelength or wavelengths to emerge from and enter the sample compartment to the same extent.

[0023] In some embodiments the light incident wall portion and the light emerging wall portion allow light of a different wavelength, or wavelengths, to pass through. In some embodiments the light incident wall portion and the light emerging wall portion allow light of the same wavelength, or wavelengths, to pass. In some embodiments the light incident wall portion and the light emerging wall portion allow the passage of light to a different extent. In some embodiments the light incident wall portion and the light emerging wall portion allow the passage of light to the same extent. In one embodiment the light incident wall portion and the light emerging wall portion allow light of the same wavelength, or wavelengths, to pass to the same extent. As an illustrative example the light incident wall portion and the light emerging wall portion may be transparent or at least essentially transparent in the range of visible light. In one embodiment the light incident wall portion and the light emerging wall portion are at least essentially transparent. In another embodiment, the degree to which the light incident wall portion and/or the light emerging wall portion allow light to pass through, changes along the respective wall portion in a selected direction, for example along the width or the height of the cuvette. As an illustrative example, the transmission properties of a respective wall portion may gradually or step-wise change from transparent to opaque from one end of a respective wall portion to another end. [0024] Examples of suitable material for the light incident wall portion and the light emerging wall portion include, but are not limited to, glass, quartz and plastic material. Suitable plastic materials for the construction of the light incident wall portion and the light emerging wall portion include, but are not limited to, polymethylmeacrylates (e.g. polymethylmethacrylate (PMMA) or carbazole based methacrylates and dimethacrylates), polystyrene, polycarbonate, and polycyclic olefins. A further illustrative example of a material that is additionally suitable for the generation of a wall portion that allows light to pass only to a certain extent is fluoro-ethylen-propylen (FEP). The material of the light incident wall portion and the light emerging wall portion may be selected independent from one another. In one embodiment the material of the light incident wall portion and the light emerging wall portion is identical.

[0025] As mentioned above, the distance between the points where the light enters and leaves the medium of a sample in a cuvette is called the optical pathlength. The optical pathlength of a cuvette according to the present invention is thus defined by the distance between the points where the light enters and where the light leaves the sample compartment. A point where light enters the sample compartment is located within the light incident wall portion, while a point where light leaves the sample compartment is located within the light emerging wall portion. Both the points were light enters and where light leaves the sample compartment are furthermore located within a surface area of the respective wall portion that faces the interior of the sample compartment in that it is able to contact fluid filled therein.

[0026] A conventional cuvette consists of two pairs of opposing lateral walls, as depicted in Fig. 3. As the four lateral walls of a conventional cuvette are oriented orthogonally with respect to each other, the distance between the walls is constant along the length, the width or any other dimension of the cuvette. Either one pair (Fig. 3B - Fig. 3E) or both pairs (Fig. 3A) of the lateral walls of a conventional cuvette are transparent. In order to reduce the sample volume consumed for optical measurements, the inner space of conventional cuvettes has been reduced by shortening the inner distance between non-transparent lateral walls. A typical conventional semi-micro cuvette (cf. Figures 3C and 3D) has standard cross sectional dimensions of 4 mm x 10 mm. The cross section of a micro cuvette (cf. Fig. 3E) has a length of 10 mm and a width of 2 mm or below. The constant length of the optical path in the inner space of a conventional cuvette is therefore generally the standard optical pathlength of 10 mm.

[0027] In contrast to the constant optical pathlength of a conventional cuvette, a cuvette of the present invention has at least two different optical pathlengths. This is due to the fact that the sample compartment of a cuvette of the present invention is designed such that it has at least two different optical pathlengths between the light incident wall portion and the light emerging wall portion. Thus - and regardless of the selected position of the cuvette - light that enters the sample compartment of a cuvette of the present inventions has at least two different pathlengths between the light incident wall portion and the light emerging wall portion. Accordingly, the sample compartment of the cuvette of the present invention has generally at least two different distances between the light incident wall portion and the light emerging wall portion. As a general illustrative example, Fig. 4A shows light (arrows) illuminating a cuvette of the invention from the right hand side. Accordingly, light enters the sample compartment from the right and emerges on the left hand side. As can be seen, the optical pathlength in immediate vicinity of the base of the sample compartment, i.e. close to the bottom, is several fold longer than the optical pathlength in immediate vicinity to the inlet of the sample compartment, i.e. close to the top.

[0028] The distance between the light incident wall portion and the light emerging wall portion may vary in any direction, including in a plurality of directions. Accordingly, the circumferential wall, including at least one of the two respective wall portions (i.e. the light incident wall portion and/or the light emerging wall portion), may include sections of any geometry. It may for example include at least one of a recess, a dent, a bulge, a step, a ledge, an extrusion, or any combination thereof.

[0029] In some embodiments the sample compartment is designed such that it has a plurality of optical pathlengths between the two respective wall portions. The sample compartment may for instance have a plurality of discrete optical pathlengths. As an illustrative example, at least one of the respective wall portions may be a stepped wall. Figures IF and IG depict two illustrative embodiments, where one wall portion has multiple perpendicular steps (cf. also Fig. 4C). Figure IE depicts a further illustrative embodiment, where both wall portions have multiple perpendicular steps. Each of these steps defines a section of a wall portion with a different distance to the other one of the two wall portions, which defines a discrete optical pathlength. [0030] In other embodiments where the sample compartment is designed such that it has a plurality of optical pathlengths between the two respective wall portions, the optical pathlenght may gradually change along any section of one of the wall portions. It may change in any selected direction, for instance along the height and/or the width of the cuvette. As an example, the sample compartment may have a continuously changing optical pathlength between at least a section of the light incident wall portion and a corresponding section of the light emerging wall portion along the height and/or along the width of the cuvette. In one embodiment the optical pathlength between the two wall portions continuously changes along the entire height and/or along the entire width of the cuvette. Figures IA and 1C depict two illustrative embodiments in which one of the two respective wall portions includes an inclined section. The optical pathlength between the inclined section of the wall portion (depicted on the right hand side in Fig. 1C) and the corresponding section of the other wall portion continuously changes along the height of the cuvette. Figure ID depicts a further illustrative embodiment in which two respective wall portions include an inclined section. Similar to the embodiment depicted in Fig. 1C, the optical pathlength between the two inclined sections of the two wall portions continuously changes along the height of the cuvette.

[0031] Figures IH and II depict two further embodiments where one of the two wall portions is a stepped wall. Contrary to e.g. Fig. IG, one edge of a respective step is however rounded. Along the height of the cuvette the rounded edge thus defines a section of the respective wall portion, in which the distance to the other wall portion gradually changes. The optical pathlength between the respective section of the wall portion and the corresponding section of the other wall portion therefore continuously changes.

[0032] In some embodiments where the sample compartment is designed such that it has a plurality of optical pathlengths between the two respective wall portions, at least one of the wall portions, i.e. the light incident wall portion and/or the light emerging wall portion, is a curved wall. In some embodiments a section of such a wall portion is a curved wall. In some embodiments the curved wall is reflected in the cross section of the sample compartment in the plane parallel to the base. At a respective wall portion, or a respective section of a wall portion, the cross section of the sample compartment - in the plane parallel to the base - may for instance be of oval shape, have the shape of a circle or of a semi-circle (cf. e.g. Fig. IL). In some embodiments the curved wall is reflected in the cross section of the sample compartment in the plane that is perpendicular to the base. At a respective wall portion, or a respective section of a wall portion the cross section in the plane that is perpendicular to the base may e.g. be of oval, circular, semi-circular etc. shape. The cross section of the sample compartment in the plane parallel to the base may in some of these embodiments not be effected by the curved wall, and for instance be of the shape of a square, a rectangle, a triangle, or any octahedron.

[0033] Figure IB depicts an embodiment of a flask-shaped cuvette. In this embodiment the sample compartment is designed such that it has a continuously changing optical pathlength across the sample compartment along the width of the cuvette. In addition thereto, at the central section of the circumferential wall, which corresponds to the region of a flask where the neck meets the body, the optical pathlength continuously changes along the height of the cuvette. Any portion of the circumferential wall of the sample compartment of the cuvette shown in Fig. IB may be selected as a light incident wall portion, provided that the selected wall portion allows light to pass through. Likewise any wall portion may be selected as a light emerging wall portion. It should however be noted that the light incident wall portion and the light emerging wall portion cannot be selected independent from one another. The direction of a light beam addressed at the cuvette defines the light incident wall portion. Depending on the size of the light beam, maximally half of the circumferential wall, for instance the wall portion visible to the beholder in Fig. IB, may serve as the light incident wall portion. The corresponding wall portion where the light beam emerges from the cuvette, for instance the wall portion invisible to the beholder in Fig. IB, may serve as the light emerging wall portion.

[0034] In some embodiments where the sample compartment is designed such that it has a plurality of optical pathlengths between the two respective wall portions, at least one of the two wall portions may be a straight wall. One wall portion, the light incident or the light emerging wall portion, may for instance be a curved or a stepped wall, while the other wall portion is a straight wall. In the embodiments depicted in e.g. Fig. IG or IH, one of the two wall portions is a stepped wall and the other wall portion is a straight wall. In the embodiments depicted in e.g. Fig. IA or IL, one of the two wall portions is a curved wall and the other wall portion is a straight wall.

[0035] In some embodiments the light incident wall portion and the light emerging wall portion are at least substantially parallel to each other. Fig. 4 depicts four embodiments, in which the light incident wall portion is indicated by arrows symbolizing light that illuminates the cuvette. Accordingly, the light incident wall portions of the depicted cuvettes are located on the right hand side, and the light emerging wall portions on the left hand side, seen from the beholder of the figure. The respective walls are in these cases parallel to each other.

[0036] As will be apparent from the above and the appending Figures, a cuvette of the invention may include more than one light incident wall portion and/or more than one light emerging wall portion. A plurality of light incident wall portions may for example share a common light emerging wall portion and vice versa. As an illustrative example, a cuvette may include multiple pairs of corresponding light incident wall portions and light emerging wall portions. Any number of these corresponding wall portions may for instance include a curved wall, a stepped wall or a straight wall. Any number of these corresponding wall portions may furthermore be at least substantially parallel to each other. Any of a plurality of such wall portions may for example include at least one of a recess, a dent, a bulge, a step, a ledge, an extrusion, or any combination thereof.

[0037] The cuvette of the invention may also include wall portions that do not allow light of a certain or any selected wavelength or wavelengths to pass. Such wall portions may for instance be included in the cuvette to prevent light originating from undesired light sources, for instance background light, from entering the sample compartment. The outer region of the cuvette may also include additional elements such as feet, a surface that is adapted for secure gripping of the cuvette, or scale markings indicating the volume corresponding to a certain fill height in the sample compartment.

[0038] Data obtained from detecting light that has passed different optical pathlengths of the sample compartment of the cuvette may be used for averaging in accordance with the Lambert-Beer law (supra). Particularly in embodiments where a plurality of different optical pathlengths is analysed, it may be desired to apply an automated analysis method, for example a software. Where a cuvette with a curved wall portion such as e.g. depicted in Fig. 4 B is used for the first time at a defined position with respect to a light source, it may be required to calibrate data analysis with e.g. samples of known properties. [0039] Averaging of data obtained from measurements at different optical pathlengths of the cuvette of the invention may furthermore be advantageous in cases, where the composition of a sample changes during a measurement or between a plurality of consecutive measurements. As an illustrative example, the course of a chemical reaction or the growth of a microorganism in a sample may be measured within a selected time frame. The respective chemical reaction or growth of a microorganism may proceed at different rates within the sample. This may for instance be due to inhomogeneities of sample components such as reactants, catalysts, nutrition components or growth factors. In addition to the possibility of averaging, the cuvette of the present invention may also be used to determine fluctuations and deviations in the composition of a sample, including the determination of standard deviation. Such data may for instance be useful to reveal micro-inhomogeneities of a sample or indicate the low solubility of a sample component. Accordingly, the cuvette of the invention may for instance be useful in observing the course of e.g. aging of a sample or in determining its suitability for a desired use. It may thus be desired to use the cuvette of the invention for instance for quality control purposes. [0040] Apart from data analysis by averaging, a profile of obtained data may be generated in order to identify certain pathlengths which are more or which are less error prone than others, or which provide a particularly high or low signal to noise ratio. Again it may be desired to apply calibration, for instance by adding matter of known optical properties, in order to identify pathlengths that provide particularly reliable data for a selected sample or for a selected type of sample. In this way data analysis may be further optimized and errors in measurement further reduced. It will be appreciated by the skilled artisan that the usage of a cuvette according to the present invention thus not only increases the signal to noise ratio and reduces errors in measurement, but furthermore can be used to optimize the optical pathlength of a conventional cuvette for a selected sample. [0041] The present invention also provides a cuvette holder. The cuvette holder is capable of accommodating a cuvette as described above. The cuvette holder is designed to match the outer shape of the cuvette. The cuvette holder may for example match the shape of a cuvette with a circumferential wall that includes at least one of a recess, a dent, a bulge or a step. In some embodiments the cuvette holder may be adapted to match a cuvette with a light incident wall portion and a light emerging wall portion, at least one of which is a curved wall. In some embodiments the cuvette holder is designed to match one defined cuvette shape. A respective cuvette holder is for example depicted in the right part of Fig. 5D. In other embodiments the cuvette holder is designed to match a plurality of cuvette shapes, as for instance depicted in the lower part of Fig. 5D.

[0042] In some embodiments the cuvette holder includes a light incident wall portion and a light emerging wall portion. These two wall portions are designed to overlap at least a part of the light incident wall portion and at least a part of the light emerging wall portion of the circumferential wall of the cuvette, respectively, when the cuvette is accommodated by the cuvette holder. The light incident wall portion and/or the light emerging wall portion of the cuvette holder thus typically have the function of a window. The design of the cuvette holder allows light to enter into and emerge from the sample compartment of the cuvette via its light incident wall portion and its light emerging wall portion, respectively. The two wall portions may overlap any part of the two wall portions of the circumferential wall of the cuvette as long as light can pass through the two wall portions of the cuvette. Accordingly, the light entering the cuvette via the cuvette holder has typically at least two different optical pathlengths between the first and the light incident wall portion and the light emerging wall portion. In some embodiments the light incident wall portion and the light emerging wall portion of the cuvette holder fully overlap the two respective wall portions of the circumferential wall of the sample compartment of the cuvette. The two wall portions of the cuvette holder may have the same or different degrees of transmittance for light. The may allow light of any selected wavelength or range of wavelengths to pass through. In one embodiment both wall portions are at least essentially transparent. The two wall portions of the cuvette holder may be of identical, similar or different material. Examples of a suitable material include, but are not limited to, glass, quartz and plastic material (cf. also above).

[0043] In some embodiments the cuvette holder includes an opening designed to overlap at least a part of the light incident wall portion of the circumferential wall of the sample compartment of the cuvette and/or an opening designed to overlap at least a part of the light emerging wall portion of the respective circumferential wall of the cuvette. The cuvette holder may for instance include an opening that overlaps at least a part of the respective light incident wall portion of the sample compartment of the cuvette, once a cuvette is inserted into the cuvette holder. In some embodiments the opening is designed to fully overlap the respective light incident wall portion of the sample compartment of the cuvette. In some embodiments the cuvette holder further contains an opening that overlaps at least a part of the respective light emerging wall portion of the sample compartment of the cuvette, once a cuvette is inserted into the cuvette holder. In some of these embodiments the cuvette holder contains a light emerging wall portion that overlaps at least a part of the respective light emerging wall portion of the sample compartment of the cuvette, once a cuvette is inserted into the cuvette holder (see above).

[0044] In further embodiments the cuvette holder may for example include an opening that overlaps at least a part of the respective light emerging wall portion of the sample compartment of the cuvette, once a cuvette is inserted into the cuvette holder. In some of these embodiments the cuvette holder contains a light incident wall portion that overlaps at least a part of the respective light incident wall portion of the sample compartment of the cuvette, once a cuvette is inserted into the cuvette holder (see above). In some embodiments the opening is designed to fully overlap the respective light emerging wall portion of the sample compartment of the cuvette. In yet further embodiments the cuvette holder includes two openings. The first opening is designed to overlap at least a part of the light incident wall portion of the circumferential wall of the sample compartment of the cuvette, when the cuvette is accommodated by the cuvette holder. The second opening is designed to overlap at least a part of the light emerging wall portion of the circumferential wall of the sample compartment of the cuvette, when the cuvette is accommodated by the cuvette holder. In one embodiment both openings are designed to fully overlap the corresponding light incident and light emerging wall portion of the cuvette, respectively, when the cuvette is accommodated by the cuvette holder. In all these embodiments the cuvette holder is designed such that light can enter into and emerge from the sample compartment of the cuvette via the first opening or respective wall portion of the cuvette holder and the light incident wall portion of the cuvette, and via the second opening or respective wall portion of the cuvette holder and the light emerging wall portion of the cuvette, respectively.

[0045] In some embodiments the cuvette holder includes more than one opening, light incident, and/or light emerging wall portions that are designed to overlap (fully or partly) with a plurality of respective wall portions of a cuvette. In some embodiments the cuvette holder includes a plurality of such openings or wall portions that are designed to overlap with a single wall portion of a cuvette of the invention. In the latter case typically each of the openings or respective wall portions of the cuvette holder overlaps with a section of the light incident or light emerging wall portion of the cuvette. Such a cuvette holder design may for example be selected for a cuvette with e.g. a wall portion of a curved wall, in order to separate defined subranges of optical pathlengths, for instance for calibration purposes.

[0046] The invention further provides an apparatus for measuring the absorption and/or emission of light in a fluid sample. The apparatus may for instance include a light source and a detector. A light source of the apparatus may provide light of any intensity and any wavelength or wavelengths. It may provide polarized light, including circularly polarized light. The apparatus may be designed to be used for spectroscopy, i.e. the dependence of physical quantities on frequency, including fluorescence spectroscopy and/or circular dichroism (CD) spectroscopy. The latter determines differences in the absorption of left-handed polarized light versus right-handed polarized light. Fluorescence spectroscopy (also called fluorometry) is based on the property of some molecules to emit light upon excitation by light of a certain wavelength. The apparatus may also be designed to be used to detect luminescence from a fluid sample. The detection of luminescence does generally not require irradiation of the sample in order to initiate light emission.

[0047] The apparatus is capable of accommodating a cuvette as described above, including a plurality of respective cuvettes. In some embodiments the apparatus includes a cuvette holder as described above, which is capable of accommodating a cuvette as described above. In some embodiments the apparatus includes a cuvette as described above. As an illustrative example, a plurality of cuvettes may be included in a sample holding unit as depicted in Fig. 6. The cuvette may for instance be removable from the apparatus, for instance from a cuvette holder included in the apparatus. The apparatus may include or be connected to any additional element, device or apparatus. The apparatus may for example include a control unit (Fig. 6, Fig. 8) or a data storage unit (Fig. 7). As a further example, the apparatus may include a data processing unit or be connectable or connected to a computer. The apparatus may for instance be designed to collect, process, store and display data obtained using a cuvette of the invention. Figure 9A depicts an illustrative example of a user interface for data analysis,

Figures 9B and 9C two illustrative examples of the display of respective data.

[0048] The apparatus may be designed for measurements of samples of any origin, as long as the sample is or can be included in a fluid. A respective sample may for instance, but not limited to, be derived from humans, animals, plants, bacteria, viruses, spores, fungi, or protozoae, or from organic or inorganic materials of synthetic or biological origin. Accordingly, any of the following samples selected from, but not limited to, at least one of a soil sample, an air sample, an environmental sample, a cell culture sample, a bone marrow sample, a rainfall sample, a fallout sample, a sewage sample, a ground water sample, an abrasion sample, an archaeological sample, a food sample, a blood sample, a serum sample, a plasma sample, an urine sample, a stool sample, a semen sample, a lymphatic fluid sample, a cerebrospinal fluid sample, a nasopharyngeal wash sample, a sputum sample, a mouth swab sample, a throat swab sample, a nasal swab sample, a bronchoalveolar lavage sample, a bronchial secretion sample, a milk sample, an amniotic fluid sample, a biopsy sample, a cancer sample, a tumour sample, a tissue sample, a cell sample, a cell culture sample, a cell lysate sample, a virus culture sample, a nail sample, a hair sample, a skin sample, a forensic sample, an infection sample, a nosocomial infection sample, a production sample, a drug preparation sample, a biological molecule production sample, a protein preparation sample, a lipid preparation sample, a carbohydrate preparation sample, a space sample, an extraterrestrial sample or any combination thereof may be processed in the method. Where desired, a respective sample may have been preprocessed to any degree. As an illustrative example, a tissue sample may have been digested, homogenised or centrifuged prior to being used with the device of the present invention. The sample may furthermore have been prepared in form of a fluid, such as a solution. Examples include, but are not limited to, a solution or a slurry of a nucleotide, a polynucleotide, a nucleic acid, a peptide, a polypeptide, an amino acid, a protein, a synthetic polymer, a biochemical composition, an organic chemical composition, an inorganic chemical composition, a metal, a lipid, a carbohydrate, a combinatory chemistry product, a drug candidate molecule, a drug molecule, a drug metabolite or of any combinations thereof. Further examples include, but are not limited to, a suspension of a metal, a suspension of metal alloy, and a solution of a metal ion or any combination thereof, as well as a suspension of a cell, a virus, a microorganism, a pathogen, a radioactive compound or of any combinations thereof. It is understood that a sample may furthermore include any combination of the aforementioned examples. [0049] As explained above, the cuvette of the invention may be of any desired dimensions. Where the sample is of small volume, a correspondingly small apparatus may likewise be selected. In some embodiments the apparatus may be portable. This may allow for the analysis of a sample directly at the site of its collection, thus providing for example a fast diagnosis, quality or damage assessment. [0050] The invention will be better understood when considered in conjunction with the non-limiting examples and the accompanying drawings, in which:

[0051] Figure 1 depicts exemplary embodiments of a cuvette according to the present invention. A circumferential wall (3) and a base (2) define a sample compartment (see also Fig. 4), which includes an inlet (4). The circumferential wall includes a light incident wall portion and a light emerging wall portion (one on the left, one on the right hand side of the shown cuvettes). There are at least two different distances between the two wall portions. At least one of the two wall portions is a curved wall (A, B, H, I, K, L, M), a stepped wall (E, F, G, H I), or at least inclined with respect to the other wall portion (A, B, C, D, K).

[0052] Figure 2 shows an embodiment of a cuvette of the invention, in which the inlet is an opening and the cuvette includes a removable seal (6) for the opening.

[0053] Figure 3 depicts conventional cuvettes. Four lateral walls (3) and a base (2) define a sample compartment, which includes an inlet (4), which is an opening. All four (A) or two opposite lateral walls (B, C, D, E) have at least a degree of transparency. The distance between the respective walls is constant within one cuvette. Arrows indicate the direction of light irradiating the cuvette.

[0054] Figure 4 depicts exemplary embodiments of a cuvette according to the present invention. Arrows indicate the direction of light irradiating the cuvette. A sample compartment (5) of the cuvette, which includes an inlet (4), is defined by a circumferential wall (3) and a base (2).

[0055] Figure 5 depicts exemplary embodiments of a cuvette holder according to the present invention. An arrow indicates how a corresponding cuvette with a wall portion that is a curved wall (A, B) or a stepped wall (C, D) can be inserted into the cuvette holder. [0056] Figure 6 shows a cuvette (1) inserted into a sample holding unit of an apparatus according to the present invention. The apparatus includes a control unit (11).

[0057] Figure 7 depicts an example of a data storage component of an apparatus of the invention, which is a memory reader unit (10).

[0058] Figure 8 shows an exemplary apparatus of the invention, which includes a sample holding unit (12), a control unit (11), a memory storage moduel (14) and a power adaptor (13).

[0059] Figure 9A depicts a control window of data analysis software used in conjunction with an apparatus of the invention. Data analysis can be adjusted according to sample origin. [0060] Figure 9B shows a further control window of data analysis software depicting stored data after analysis.

[0061] Figure 9C shows another control window of data analysis software, displaying graphically data obtained using a cuvette of the invention, included in the apparatus.

EXAMPLES

Example 1; Cuvette Fabrication

[0062] The present example illustrates a method of fabricating a cuvette according to the present invention. The embodiment fabricated in this example is depicted in Fig. 2.

[0063] Polymethylmethacrylate (amorphous, density 1.16 g/cm³, melting point 100 ⁰C) was selected as material for the cuvette, as it is a clear transparent plastic that transmits more light than glass and allows UV light down to 300 nm and infrared light up to 2800 nm to pass. The cuvette was formed in a mould of 718 steel (36 hardness) by injection moulding using a conventional injection unit.

[0064] Briefly, a two-plate mold construction is employed, using a submarine (tunnel) gate, which is automatically trimmed from the cavity. In this gate the opening from the runner into the mold cavity is located below the parting line. An angled, tapered tunnel is machined from the end of the runner to the cavity, just below the parting line. As the parts and runners are ejected, the gate is sheared at the part. As for other submarine gates, the tunnel can be located either in the moving mould half or in the fixed half. A sub-gate may furthermore be located into the side of an ejector pin on the non-visible side of the part when appearance is important. Degating is performed in an automated manner. A good taper of the tunnel is required for degating, which furthermore must be free to bend.

[0065] A cap for the cuvette was formed from polypropylene homopolymer (crystalline, density 0.90 g/cm³, melting point 170 ⁰C).

Example 2: Absorbance Measurement for the Detection of Porcine Pathogens

[0066] The present example illustrates an exemplary use of a cuvette and an apparatus according to the present invention.

[0067] The diagnostic system used in the present example is a rapid diagnostic system that is able to detect and monitor trends of three main targets: (a) feed quality/safety, (b) water quality/safety, and (c) porcine-related pathogens, as indicators of impending porcine-related problems. The unique feature of the diagnostic system is its ability to allow for regular on-site deterministic tracking and analysis of these target indicators. This provides on the spot information for impending outbreak of disease on the farm, porcine growth optimization, etc. The diagnostic system also features low cost, high specificity and is easy to use.

[0068] For a large majority of pig farms, porcine diseases are currently determined pathologically, or serologically using laboratory-based ELISA and culture techniques. Farms in the ASEAN region that conform to regulatory requirements send their specimens to authorized testing laboratories abroad (e.g. Korea, Japan, and Germany). The test results typically take 3 weeks. Such long waiting times are intimately associated with compounding problem proliferation on the farm that prospectively results in significant losses in damages. Sending specimens abroad has other disadvantages as well. Apart from slow reaction to containment of an outbreak, the high dependence on sample integrity before and after freight to yield accurate results may be in question (i.e. false-negative results).

[0069] With respect to early detection of porcine disease, farmers are limited to observing the porcine behaviour, signs and symptoms and through autopsy of a sacrifice

(especially if there is a vet on site). In order to provide early and rapid detection of porcine disease, the introduction of rapid, portable, simple to use biosensors on the farm was deemed to be of substantial impact for farming in general.

Diagnostic System

[0070] The diagnostic system includes three major components: the hardware system, the cuvette, and the software (MicroSen, ASensor, Singapore). The hardware system includes a sample holding unit (including a cuvette holder), a control unit, a memory storage unit and a memory reader unit (shown in e.g. Figure 8).

[0071] The sample-holding unit acts as a portable incubator chamber. Since most microorganisms' optimal growth occurs at the temperature of the human body, the sample- holding unit is set to operate at 35-37 ⁰C. Where desired, the sample-holding unit can be adapted to operate at any temperature ranging from 25-50 ⁰C. The sample-holding unit of the apparatus used also functions as a spectrometer, which only operates at 547 nm. It has a maximum capacity of testing 8 samples per test.

[0072] The control unit is connected to the sample-holding unit. Its function is to control the temperature of the incubator, intensity of the light source and saves testing data in a certain time interval. The control unit measures the absorbance in terms of Counts. It has a measurement range of 0-7000 counts (1 count is equivalent to 0.305178 mV).

[0073] Data is stored in terms of absorbance counts in the memory storage unit. The data in the memory storage unit could be uploaded to a personal computer using the memory reader unit and the MICROSen software.

The MICROSen Software [0074] MICROSen (snapshots are shown in Figure 9) acts as a database to store and analyze a group of collected data. MICROSen is able to keep track and monitor a series of cumulative data over a period of time. Users are able to sort out the data results based on type of samples, location where the samples were taken from, date, and mortality rate. In combination with the hardware, MICROSen provides a rapid monitoring to show and detect any level changes of microbial loads in water and feed. It is also able to determine the APP level in the tested sample. Results of the microorganism loadings are presented in graphs that are categories into three levels (i.e. Low, Medium, High) (cf. Figure 9C).

[0075] In addition, a program "MEMZip" is incorporated in the software to allow transferring of raw data to MICROSen for data analysis. For security purposes, MICROSen has the feature of an administrator control to create password protect function for all account users.

A recovery system is also modeled in the software to create a backup and restore function. This software has an expandable function to accommodate any future expansion of farm units.

[0076] The detection system targets three microbiological agent related to porcine disease. The three targets are: (1) total bacteria loading in water, (2) total fungal loading in feed, and (3) Actinobacillus pleuropneumoniae (APP). These sensors are designed to be user friendly, easy to use and not laborious. Software capable of processing output data from the sensor is provided with the detection system to handle data analysis. Each target, in the present case APP, has its own specific algorithm that can be incorporated in the software. The respective algorithm was developed based on the absorbance change occurring as a result of bacteria growth cultured in the cuvette in customized culture media.

Total Bacteria Sensor (Hydrobacter)

[0077] The Bacteria Sensor is able to indicate the total amount of bacteria loading that is present in the collected water samples. The detection time is from 4 to 8 hours. The testing only requires one handling step, which is to transfer the collected water to the cuvette.

Fungus Tester (Mycespoter) [0078] The Fungus Tester is designed to detect the presence of fast growing fungus, e.g. Aspergillus sp., which is present in the feed. The detection time is from 10 to 24 hours. The operation of the sensor is a two step process.

APP Tester (ActBacter) [0079] Actinobacillus pleuropneumoniae (APP) is an important pig pathogen causing swine pleuropneumonia, a highly contagious respiratory infection. APP infections impact swine production due to mortality and medical costs accompanying an acute outbreak. The APP Tester is used as a fast screening instrument to know if a pig is infected by APP. Samples required are swab samples from the pig's nostrils. With the specific developed algorithm, the sensor differentiates APP growth from other bacteria that is present in the collected swab samples. The detection time is from 8 to 10 hours. The operation of the sensor is a two step process.

[0080] The diagnostic system is used together with liquid culture media, which are kept at 2 to 8 ⁰C for storage. Before use the media are allowed to warm to room temperature. Samples are collected and transferred into the cuvette at a volume that is sufficient to allow the sample to fully contact all light incident and light emerging wall portions desired to be used. The cuvette is then sealed by the respective cap and the cuvette inverted up and down five times. Thereafter the cuvette is inserted into the cuvette holder of the sample holding unit, irradiated at 547 nm, and recorded data stored in the memory storage unit. At any desired point in time the memory storage unit is then connected to a personal computer and the data analysed using the MICROSen software.

[0081] The invention has been described broadly and generic herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0082] Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, the skilled artisan will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

ClaimsWhat is claimed is:

1. A cuvette comprising a sample compartment with an inlet, the sample compartment being defined by a circumferential wall and a base, the circumferential wall having a light incident wall portion and a light emerging wall portion, the light emerging wall portion being opposite to said light incident wall portion, wherein said sample compartment is designed such that it has at least two different optical pathlengths between the light incident wall portion and the light emerging wall portion.

2. The cuvette of claim 1, wherein said sample compartment is designed such that it has a plurality of discrete optical pathlengths between the light incident wall portion and the light emerging wall portion.

3. The cuvette of claim 1, wherein said sample compartment is designed such that it has a continuously changing optical pathlength between at least a section of the light incident wall portion and a corresponding section of the light emerging wall portion along the height and/or width of the cuvette.

4. The cuvette of any one of claims 1 - 3, wherein at least one of said wall portions is a curved wall.

5. The cuvette of any one of claims 1 - 4, wherein at least one of said wall portions is a stepped wall.

6. The cuvette of claims 4 or 5, wherein the other one of said wall portions is a straight wall.

7. The cuvette of any one of claims 1 - 6, wherein the said two wall portions are at least substantially parallel to each other.

8. The cuvette of any one of claims 1 - 7, wherein at least one of said wall portions comprises at least one of a recess, a dent, a bulge, a step, a ledge, an extrusion, and any combination thereof.

9. The cuvette of claim 8, wherein said light incident wall portion and said light emerging wall portion are at least essentially transparent.

10. The cuvette of any one of claims 1 - 9, wherein said inlet is located on a top of the sample compartment being at least essentially opposite to said base.

11. The cuvette of claim 10, wherein said inlet is an opening.

12. The cuvette of claim 11, further comprising a removable seal for said opening.

13. A cuvette holder capable of accommodating a cuvette according to any one of claims 1 -

12, said cuvette holder being designed to match the outer shape of said cuvette.

14. The cuvette holder of claim 13, comprising a light incident wall portion and a light emerging wall portion designed to overlap at least a part of said light incident wall portion and at least a part of said light emerging wall portion of the circumferential wall of the cuvette, respectively, when the cuvette is accommodated by said cuvette holder, so that light can enter into and emerge from the sample compartment of the cuvette via said light incident wall portions and said light emerging wall portions, respectively.

15. The cuvette holder of claim 14, wherein the light incident wall portion and the light emerging wall portion of the cuvette holder fully overlap the light incident wall portion of the sample compartment of the cuvette and the light emerging wall portion of the circumferential wall of the cuvette, respectively.

16. The cuvette holder of claim 13, comprising a first opening designed to overlap at least a part of said light incident wall portion of said sample compartment of the cuvette and a second opening designed to overlap at least a part of said light emerging wall portion of said sample compartment of the cuvette, respectively, when the cuvette is accommodated by said cuvette holder, so that light can enter into and emerge from the sample compartment of the cuvette via said first opening of the cuvette holder and the light incident wall portion of the cuvette, and via said second opening of the cuvette holder and the light emerging wall portion of the cuvette, respectively.

17. The cuvette holder of claim 16, wherein said first and said second openings are designed to fully overlap said light incident wall portion and said light emerging wall portion of the sample compartment of the cuvette, respectively.

18. An apparatus for measuring the absorption and/or emission of light in a fluid sample capable of accommodating a cuvette according to any one of claims 1 — 12.

19. The apparatus of claim 18, comprising a cuvette holder according to any one of claims 13

- 17.

20. The apparatus of claims 18 or 19, comprising a cuvette according to any one of claims 1 -

12.