WO2014072053A1

WO2014072053A1 - Microcuvette for biochemical assays

Info

Publication number: WO2014072053A1
Application number: PCT/EP2013/003343
Authority: WO
Inventors: Piotr KNAP; Piotr Garstecki; Marcin Izydorzak; Magdalena SULIMA; Adam WARCHULSKI
Original assignee: Pz Cormay S.A.
Priority date: 2012-11-07
Filing date: 2013-11-07
Publication date: 2014-05-15
Also published as: PL401491A1

Abstract

The invention relates to a microcuvette, in particular for biochemical assays, allowing for the measuring of analyte concentration in a sample, comprising a chamber for the sample, characterised in that it comprises a first chamber (1) and a second chamber (2), whereas the second chamber (2) is covered by a rotatable sleeve, the lateral surface of which separates the first chamber (1) and the second chamber (2), and the lateral surface of the sleeve comprises an opening (6) that can be so positioned, by rotating the sleeve, that it provides connection between the first chamber (1) and the second chamber (2).

Description

Microcuvette for biochemical assays

The subject matter of the invention is a microcuvette, in particular for biochemical assays, allowing for the measuring of analyte concentration in a sample.

The present invention relates to a microcuvette (microvessel) for carrying out reactions characteristic for individual analytes occurring in body fluids. The analytes in microcuvettes are detected using spectrophotometric methods (absorbance measurements).

In standard applications, polystyrene cuvettes with 1 cm_z or possibly 5 mm^ optical paths are used (see, Hans-Ulrich Wittchen "Reliability and validity studies of the WHO- Composite International Diagnostic Interview (CIDI): A critical review" 1993). These vessels are commercially available in a range of volumes. The volumes are usually in the order of milliliters^ which translates into quite a high consumption of reagents used in measurements. Polystyrene cuvettes are sufficiently transparent for measurements in the wavelength range of from 340 nm to 900 nm.

Table 1 below lists biochemical assays together with reagent-serum volume ratios commercially used in reactions (abbreviations: S - volume of serum, VI - corresponding volume of the Rl reagent used as a standard practice, V2 - corresponding volume of the R2 reagent where used as a standard practice). Depending on the parameter to be determined in an assay, one (Rl) or two (Rl and R2) reagent solutions are used. In some cases, partitioning necessary reactants into the two reagent solutions results in an improved reagent stability . Most parameters are labelled by reagent abbreviations followed by their full names in parentheses. Table 1

tg (triglycerides) 3 180 45

Biochemistry analysers used in standard practice are advanced automated devices designed to measure the contents of various chemicals in blood. Using a set of needles and a specially designed mecha ism, a biochemistry analyser collects a serum (or plasma) sample from a tube, mixes it in a small cuvette with appropriately selected reagents, and by measuring, for example photometrically, the absorption of light by the products of a chemical reaction, the concentration of chemicals of interest in blood can be determined.

Biochemistry analysers, so constructed and capable of carrying out the above- described test procedure, are commercially available (e.g., Flexor XL analysers manufactured by ELITech), and disclosed in numerous patents and patent applications (e.g., publications JP 8211072 A, JP 10132735 A, JP 201033924 A, US 20040185549 Al, US20050014274 Al, US 4808380, US 6162399, or US20070065945 Al).

Another group of biochemistry analysers are devices using disposable discs (e.g., commercially available Abaxis Piccolo^® Xpress and Samsung IVD-A10A models), containing reagents that are necessary to carry out diagnostic reactions (existing devices use reagents in freeze-dried form), and allowing for use of the necessary biological material. The dispensing in these devices is performed with the use of centrifugal force that should assure uniform spreading out of biological material and solvent to places where the freeze-dried reagents are stored and reactions take place. The fundamental limitations for the accuracy of measurements performed with the disc technology-based analysers are related to the reproducibility of small portions of reagents in freeze-dried form and the reproducibility of dispensing. These instruments are also complex as far as their designs are concerned, and therefore quite expensive. Alternative solutions for microcuvettes are microsystems, where detection is performed in channels with cross sections of a fraction of a millimeter in size. A problem associated with this solution is the selection of a suitable, easy-to-process, transparent material that could be used to manufacture the reaction system. The tightness of such a system is also a difficult aspect. To prepare for a measurement, two immiscible phases, such as water (here: the reaction mixture) and oil are pumped into the microchannels of the system. Often the carrier phase (oil) penetrates into cavities between the glued plates of the microsystem, which consequently has a critical effect on the absorbance readout.

The inventors of the present invention have noticed that individual assays can be performed while reducing reagent volumes down to the microscale, if an appropriately designed microcuvette is used.

Therefore, according to the present invention a microcuvette is provided for performing biochemical reactions on the microscale, which is related to a low consumption of reagents for analyses. The polymer materials used to manufacture the microcuvette were selected in consideration of properties related to the position of the mixture droplets in the measurement space, and more specifically - the properties related to the interaction of the reagent with the substrate and the walls of the microcuvette. The polystyrene cuvette is wetted by oil pumped into the cuvette, and the reagent fills up precisely the measurement space as disclosed in detail in the International patent application no. PCT/EP2012 367861: "Method for determining biochemical parameters of a body fluid". This solution allows for the reduction of consumption of reagents for assays, and consequently of the price of each assay.

A microcuvette, in particular for biochemical assays, allowing for the measuring of analyte concentration in a sample, comprising a chamber for the sample, according to the invention is characterised in that it comprises the first chamber and the second chamber, and the second chamber is covered by a rotatable sleeve, the lateral surface of which separates the first chamber and the second chamber, and the lateral surface of the sleeve comprises an opening that can be so positioned by rotating the sleeve that it provides a connection between the first chamber and the second chamber. Preferably, the sleeve comprises an opening connecting the outside of the sleeve with the first chamber.

Preferably, the sleeve comprises at least one well for a tool used to rotate the sleeve.

Preferably, the sleeve is manufactured from a hydrophobic material, such as polypropylene, polyethylene, COC, PMMA.

Preferably, the microcuvette according to the present invention comprises at least two walls closing the first chamber, parallel to each other.

Preferably, the microcuvette according to the present invention is manufactured from a transparent material, preferably polystyrene, COC, PMMA.

Preferably, the microcuvette according to the present invention comprises walls that are manufactured from different material than the sleeve.

Preferably, the microcuvette according to the present invention comprises one or more openings selected from the group comprising: a) an opening connecting the outside of the microcuvette with the first chamber, preferably arranged in the bottom or in the side wall of the microcuvette; b) an opening connecting the outside of the microcuvette with the second chamber, preferably arranged in the bottom or in the side wall of the microcuvette; c) two venting openings, connecting the outside of the microcuvette with the first chamber, preferably arranged in the bottom or in the side wall of the microcuvette; d) a venting opening, connecting the outside of the microcuvette with the second chamber, preferably arranged in the bottom or in the side wall of the microcuvette.

Preferably, the microcuvette according to the present invention comprises catches for interconnecting microcuvettes.

Preferably, the microcuvette according to the present invention comprises upper framing for fastening a lid. In such a case, preferably, the microcuvette according to the present invention comprises a lid fastened to the upper framing, more preferably manufactured from the same material as the microcuvette.

In such a case, preferably, the lid comprises an opening arranged over the sleeve so that it is possible to attach a tool to the wells in the sleeve and to rotate the sleeve.

Preferably, the microcuvette according to the present invention has the dimensions 14.7 mm x 7.8 mm x 4.5 mm.

Preferably, the capacity of the first chamber is 30.5 μΙ, and the capacity of the second chamber is 7.5 μΙ.

Preferably, the volume ratio of the first chamber to the second chamber is 4:1.

Preferably, the cross section of the second chamber is triangle shaped.

Preferably, the microcuvette according to the present invention additionally comprises a third chamber, the third chamber being covered by a rotatable sleeve, the lateral surface of which separates the first chamber and the third chamber, and the lateral surface of the sleeve comprises an opening that can be so positioned by rotating the sleeve that it provides connection between the first chamber and the third chamber.

Polystyrene is a material particularly suitable for storing hydrophilic reagents. The corners of a polystyrene microcuvette are well wetted by oil and the mixture of reagent(s) with sample entirely fills up the space wherein the absorption of radiation is measured, and allows for precise detection, e.g., spectrophotometrically. The precision is directly related to the reproducibility of the optical path. The use of the microcuvette designed according to the present invention allows for substantial reduction of reagents for assays, and consequently of the price of a single assay.

The microcuvette allowing to perform intended reactions, indicating the concentration of a given analyte in a sample, is primarily characterised in that the reactions to be carried out therein will occur in a microscale, which has a beneficial effect on the reduction of the amounts of reagents consumed in the assays. In line with reduced reagent volumes, the testing consumes small samples of, e.g., plasma or serum obtained from human blood. For instance, single puncture of a fingertip can yield material for a few, an over a dozen, up to a few tens assays. The microcuvette according to the present invention is composed of one or more chambers for storing individual reagents. The mechanism allowing to separate reagents, in reactions involving multiple reagents, plays an essential role in the design of the microcuvette. The microcuvettes can be interconnected, which allows for performing a number of assays of various parameters of the sample.

Detailed description of the invention

The inventors of the present invention designed a reaction microcuvette for measurements, in particular spectrophotometric measurements, in microvolumes. The microcuvette disclosed herein is a reaction vessel for carrying out characteristic reactions to determine concentration of a given analyte in a sample. The microvessel, preferably manufactured from polystyrene, comprises two separated spaces: the first space (chamber) - 1, and the second space (chamber) - 2 (as in Fig. 1). An important element is the upper framing - 3 that is necessary to glue the microcuvette together with the lid so as to fabricate a hermetic measurement vessel. The walls of the microcuvette, in the area of the optical path, i.e., where the light beam passes the microcuvette, are parallel to each other. The light passing the microcuvette is substantially neither refracted nor scattered during the measurement. A barrier separating the reagents is a polypropylene sleeve (shown in Fig. 2). The sleeve separates the reagents (for assays involving multiple reagents; hereafter referred to as Rl and R2 reagents) used in reactions, which has a beneficial effect on the stability and durability of individual reagents. The sleeve is the cylinder shaped part with special grooves (Fig. 2). In the discussed embodiment the sleeve comprises a few essential elements:

• opening 4, used to introduce the sample into the chamber 1, wells 5a and 5b (in examples of shaping), used to attach a tool (not shown) and to rotate the sleeve to mix the Rl reagent from the chamber 1 with the R2 reagent from the chamber 2,

• opening 6, through which the R2 reagent flows into the chamber 1.

The volume of the chamber 1 and the volume of the chamber 2 can be different. The volumes are selected so as to reflect the proper ratio of reagents' volumes In reactions involving multiple reagents (e.g., the ratio of reagent volumes VI and V2, as indicated in Table 1). Usually, the reagent volume ratio, VI to V2, is 4 to 1. Other proportions are also used, for example for reagents for CRP, ferritin, or lipase assays. In the discussed embodiment, the chamber 2 of the microcuvette was designed as a triangle shaped space, which facilitates flowing of the R2 reagent to the chamber 1.

Preferably, the microcuvette is manufactured from polystyrene, because it is a polymer that is not wetted by an aqueous reagent, which is preferably surrounded by oil, e.g., hexadecane, thus supporting an appropriate setting of a droplet of a reaction mixture in the cuvette, and a polypropylene sleeve supports this effect, also because of its hydrophobic properties. The oil used also serves to seal the small space between the microcuvette and the sleeve so as to prevent contamination of one reagent with the other during storage of different reagents. Other preferred materials, indicated in the patent application P-397071, are polyethylene and Teflon. In non-limiting examples of embodiments these materials can be replaced with other hydrophobic, transparent polymers, e.g., COC (cycloolefin (co)polymer), PMMA (poly (methyl methacrylate)).

In order to be able to store reagents in the microcuvette for a long time, the microcuvette should be closed by gluing a lid on its top, preferably fabricated from the same material as the microvessel (Fig. 5). Preferably a gluing method is used here that is based on dissolving the surfaces with appropriate solvent, for polystyrene it is acetone. The microcuvette lid comprises an opening, arranged so that the sleeve can be freely rotated after gluing. A foil is glued on the lid to assure that the system is hermetically closed. To fill up the glued microcuvette, appropriately arranged openings are required (Fig. 6): the opening 8 used to introduce oil and reagent to the first chamber 1, the opening 9 used to introduce oil and reagent to the second chamber 2, the openings 10 a and 10 b for venting the system during the filling operation. The opening 8 provides connection between the outside of the microcuvette and the first chamber 1, and preferably is located in the bottom or in the side wall of the microcuvette. The opening 9 provides connection between the outside of the microcuvette and the second chamber 2, and preferably is located in the bottom or in the side wall of the microcuvette. The openings 10 a and 10 b comprise one or more from among the following openings: a) venting opening 10 a, providing connection between the outside of the microcuvette and the first chamber (1), preferably arranged in the bottom or the side wall, b) venting opening 10 b, providing connection between the outside of the microcuvette and the second chamber (2), preferably arranged in the bottom or the side wall, and c) additional venting opening 10 a , preferably arranged in the bottom or in the side wall of the microcuvette.

The venting opening 10 b may be used interchangeably with the opening 9 used for filling the second chamber 2.

Preferably, the microcuvette comprises catches 7 on both sides (Fig. 3), allowing for interconnection of microcuvettes and performing multiple successive measurements. Fig. 4 shows an example of how the microcuvettes can be interconnected using catches. In this case, the microcuvette comprises on one side a catch terminated with a cylinder shaped element, and on the other side a clamping ring with dimensions selected so that by pushing the cylinder shaped element in the clamping ring the microcuvettes can be interconnected in a durable but separable way.

The invention will now be explained in detail in a preferred embodiment, with reference to the accompanying figures, wherein:

Fig. 1 shows the measurement microcuvette without the sleeve, in which there is formed a chamber 1 for the Rl reagent and a chamber 2 for the R2 reagent, and the framing.

Fig. 2 shows the sleeve in two embodiments,

Fig. 3 shows the microcuvette with the sleeve,

Fig. 4 shows a method for interconnecting two microcuvettes,

Fig. 5 shows the shape of the lid used to seal the microcuvette, where a - shows the design, and b - the physical embodiment, Fig. 6 shows the arrangement of the openings that are necessary for the filling of the microcuvette,

Fig. 7 shows an image of physical embodiment of the microcuvette, and

Fig. 8 shows a non-limiting example of a three-reagent cuvette.

The following marking is used in the Figures: 1 - the first chamber (for the Rl reagent), 2- the second chamber (for the R2 reagent), 3 - framing, 4 - opening in the upper part of the sleeve used to introduce a sample of body fluid, e.g., serum, into the microcuvette, 5a and 5b - wells in the upper part of the sleeve for inserting the tool for rotating the sleeve, 6 - opening in the lateral surface of the sleeve, through which the R2 reagent flows into the first chamber and mixes with the Rl reagent, 7 - elements (catches) used to interconnect microcuvettes, 8 - opening in the bottom for introducing oil and the Rl reagent in the first chamber, 9 - opening in the bottom for introducing oil and the R2 reagent in the second chamber, 10 - venting openings, 11 - chamber for the Rl reagent, 12 - chamber for the R2 reagent, and 13 - chamber for the R3 reagent (inside the sleeve shown in the drawing).

Preferred embodiment of the invention

A microcuvette according to the present invention has the following dimensions: 14.7 mm x 7.8 mm x 4,5 mm. In a preferred embodiment a polystyrene cuvette is glued with a polystyrene lid with acetone. A droplet of the solvent is deposited on the lid and subsequently spreads out uniformly along the edge without flowing inside, therefore dissolving the edge of the lid only. Then the microcuvette with the sleeve is adjoined and, for better tightness, inserted in a hydraulic press for 5 min. at constant pressure 200 mbar and temperature 70°C. Before filling, the opening over the sleeve is sealed by gluing with foil. The opening in the lid is necessary to enable the mechanism, used for rotating the sleeve, to be inserted in the openings 5a, 5b (Fig. 2) after the protecting foil is punctured, and subsequently to generate rotation of the sleeve with rotational movements. The sleeve is preferably manufactured from a different material than the microcuvette, e.g., from polystyrene. This is related to the properties of the solvent used for gluing the microvessel with the lid. Acetone does not dissolve polypropylene, so there is no risk of gluing the sleeve to the lid (the sleeve must have some freedom of rotation and may not be glued to the lid). Oil and reagents are introduced into the microcuvette through appropriately adapted openings (Fig. 6), the order of operations being important - oil is introduced first to wet the walls, followed by reagent - the walls are wetted, and the reagent precisely fills up the measurement window of the microcuvette. The capacity of the chamber 1 is 30.5 μΙ, while the capacity of the chamber 2 is 7.5 μΙ. After filling, all the openings (8, 9, 10) must be tightly sealed to prevent the reagent from leaking out of the microcuvette. In this way we obtain a tight microvessel for storing reagent(s) for tests.

To perform an assay in the microcuvette so prepared, the needle, used for dispensing the fluid to be tested, punctures the protecting foil, and subsequently introduces the fluid through the opening 4 (Fig. 2) into chamber 1 (Fig. 1) filled with the Rl reagent. This is the first step of the reaction. In the case of a dual reagent system, after the first step, the sleeve should be turned by 180", to enable the reagent R2 to flow from chamber 2 to chamber 1, and subsequently, using the rotating mechanism, the sleeve should be set in rotational motions, resulting in mixing the R2 reagent with the solution present in the chamber 1 and initiating a reaction characteristic for a given reagent. For single reagent systems, chamber 2 is filled with oil, and the rotation mechanism supports only the diffusion of the tested material and the Rl reagent. Apart from providing a storage space for the Rl reagent, chamber 1 is also the chamber where the actual spectrophotometric measurement of the reaction is performed. After an appropriate period of time, characteristic for a given assay, the emerging reaction product, usually colourful, is detected by means of a measurement of absorption of light of an appropriate wavelength, in a way known to those skilled in the art.

In other preferred embodiments, the microcuvette according to the invention may comprise more reagent chambers and more sleeves. As an example, Fig. 8 shows a microcuvette designed with triple reagent systems in view, wherein 11, 12, 13 are chambers for reagents Rl, R2, R3, respectively.

Claims

1. A microcuvette, in particular for biochemical assays, allowing for the measuring of analyte concentration in a sample, comprising a chamber for the sample, characterised in that it comprises a first chamber (1) and a second chamber (2), and the second chamber (2) is covered by a rotatable sleeve, the lateral surface of which separates the first chamber (1) and the second chamber (2), and the lateral surface of the sleeve comprises an opening (6) that can be so positioned by rotating the sleeve that it provides connection between the first chamber (1) and the second chamber (2).

2. Microcuvette according to claim 1, characterised in that the sleeve comprises an opening (4) connecting the outside of the sleeve with the first chamber (1).

3. Microcuvette according to claim 1 or 2, characterised in that the sleeve comprises at least one well (5a, 5b) for a tool used to rotate the sleeve.

4. Microcuvette according to claim 1, 2 or 3, characterised in that the sleeve is manufactured from a hydrophobic material, such as polypropylene, polyethylene, COC, PMMA.

5. Microcuvette according to any of the preceding claims, characterised in that it comprises at least two walls closing the first chamber (1), parallel to each other.

6. Microcuvette according to any of the preceding claims, characterised in that it is manufactured from a transparent material, preferably polystyrene, COC, PMMA.

7. Microcuvette according to any of the preceding claims, characterised in that its walls are manufactured from different material than the sleeve.

8. Microcuvette according to any of the preceding claims, characterised in that it comprises one or more openings selected from the group comprising: a) an opening (8) connecting the outside of the microcuvette with the first chamber (1), preferably arranged in the bottom or in the side wall of the microcuvette; b) an opening (9) connecting the outside of the microcuvette with the second chamber (2), preferably arranged in the bottom or in the side wall of the microcuvette; c) two venting openings (10 a), connecting the outside of the microcuvette with the first chamber (1), preferably arranged in the bottom or in the side wall of the microcuvette; d) a venting opening (10 b), connecting the outside of the microcuvette with the second chamber (2), preferably arranged in the bottom or in the side wall of the microcuvette.

9. Microcuvette according to any of the preceding claims, characterised in that it comprises catches (7) for interconnecting microcuvettes.

10. Microcuvette according to any of the preceding claims, characterised in that it comprises upper framing (3) for fastening a lid.

11. Microcuvette according to claim 10, characterised in that it comprises a lid fastened to the upper framing (3), preferably manufactured from the same material as the microcuvette.

12. Microcuvette according to claim 11, characterised in that the lid comprises an opening arranged over the sleeve so that it is possible to attach a tool to the wells (5a, 5b) in the sleeve and to rotate the sleeve.

13. Microcuvette according to any of the preceding claims, characterised in that it has dimensions 14.7 mm x 7.8 mm x 4.5 mm.

14. Microcuvette according to any of the preceding claims, characterised in that the volume ratio of the first chamber (1) to the second chamber (2) is 4:1.

15. Microcuvette according to any of the preceding claims, characterised in that the cross section of the second chamber (2) is triangle shaped.

16. Microcuvette according to any of the preceding claims, characterised in that it additionally comprises a third chamber (13), the third chamber (13) being covered by a rotatable sleeve, the lateral surface of which separates the first chamber (11) and the third chamber (13), and the lateral surface of the sleeve comprises an opening that can be so positioned by rotating the sleeve that it provides a connection between the first chamber (11) and the third chamber (13).