WO2008119739A1

WO2008119739A1 - Improved device for sampling a fluid and detecting an analyte

Info

Publication number: WO2008119739A1
Application number: PCT/EP2008/053656
Authority: WO
Inventors: De Tim Graaf; Van Remco Maria Es; Judith Margreet Hanneke Ogink; Van Ronald Dijk
Original assignee: Dsm Ip Assets B.V.
Priority date: 2007-03-29
Filing date: 2008-03-27
Publication date: 2008-10-09
Also published as: AR065881A1

Abstract

Provided is a device for detecting an analyte in a fluid comprising a first compartment (1) having a closed end (1.1), an open end (1.2) and a test medium (1.3), and a second compartment (2) having a first open end (2.1), connected to open end (1.2), and a second open end (2.2), wherein at least parts of compartments (1) and/or (2) are made from elastic material, characterized in that a means (1.4) for decreasing the volume of compartment (1) is present.

Description

IMPROVED DEVICE FOR SAMPLING A FLUID AND DETECTING AN ANALYTE

Field of the invention The present invention relates to a device for sampling a fluid and detecting an analyte therein.

Background of the invention

Devices suitable for detecting analytes in samples are known for many years in various applications. For instance, microbiological test methods for the determination of antibacterial compounds, particularly residues of antibiotics such as cephalosporin, penicillin, tetracycline and derivatives thereof and chemotherapeutics such as sulfadiazines and similar compounds (sulfa's), in fluids such as milk, meat juice, serum and urine have been described in CA 2056581 , DE 3613794, EP 0005891 , EP 0285792, EP 0611001 , GB A 1467439 and US 4,946,777. These descriptions all deal with ready to use tests that make use of a test microorganism and will give a result by the change indicated by an indicator molecule, for instance a change of color of a pH- and/or redox-indicator. The test systems described above usually include a test medium, such as agar in which test components are present. Such test components may be a test microorganism, indicator molecules and/or nutrients, but also other components are conceivable to the person skilled in the art. In most examples of test systems known today, the test medium is contained within a container such as a tube or a well in a microtiter plate. The sample to be analyzed then needs to be brought into said container onto the test medium. Usually, said containers are sealed in order to prevent contamination and in some cases auxiliary containers with required components such as nutrients are supplied from which said required components are to be transferred to the container with the test medium.

The problem with the test systems currently distributed on the market and/or described in literature is that they require multiple operations to be performed by the user. Since any operation is subject to the occurrence of errors, a high number of operations gives rise to a high number of possible errors and thus to a relatively unreliable and inconvenient test system. For example, a given test system may require operations such as opening an ampoule, addition of nutrients, attachment of a pipette to a syringe, withdrawal of a sample with the pipette/syringe, and application of the sample to the test medium. In most cases many or all of these operations need to be performed under aseptic conditions. Yet another problem associated with the test systems currently distributed on the market and/or described in literature is that unwanted cross- contamination can easily occur.

Reducing the risk for errors in test systems by simplification of the equipment is a problem that has been addressed before. For instance, EP O 271 102 describes a sample collection device wherein a liquid-absorbing material is used to absorb the required amount of sample without having to use complicated equipment. However, said improved test systems and methods still require multiple devices and process steps, each of which introduces risks for errors. Other publications have partly addressed these problems. For instance, in US 5,833,630 a device is disclosed comprising a sample collection capillary in combination with a cuvet containing a reagent liquid. Although the device allows for very precise and reproducible sample volumes, unfortunately transportation of the sample to the reagent liquid requires the additional handling of placing a pressure cap on the device. In WO 2003/027222 a device is disclosed that can be operated by one hand and thus circumvents additional handlings. However, this latter device comprises a single pressure point for take up of sample, removal of excess sample and release of a sampling tip. In applications where release of a sampling tip is not required, for instance in applications where the analysis section does not require re-use, these single point multiple pressure handlings are both cumbersome and liable to generate errors. In WO 2006/045749 a device is disclosed that allows for single-handed sampling of precise and reproducible volumes, however this device suffers from the disadvantage that transportation of sample fluid to the analysis section is based on the force of gravity which is insufficient to overcome adhesion forces between interior device walls and sample liquid, particularly if these sample liquids are aqueous, such as is the case with body fluids like blood, milk, sweat, urine and the like. There is thus a need for an improved, single component and easy and flawless to operate integrated sampling and analysis device.

Detailed description of the invention

The terms and abbreviations given below are used throughout this disclosure and are defined as follows. The term 'drip ring' refers to a means attached to the exterior of a device and fully enclosing the device which is designed such that any fluid which is present on the outside of the device is contained within the drip ring when the device is turned upside down. The term 'elastic' refers to the characteristic of a material in that it is capable of recovering size and shape after deformation, i.e. can be easily stretched or expanded and resuming its former shape. In the context of the present invention, elastic material refers to materials that are elastic already under relatively low pressures, i.e. those that can be easily applied by the human hand or finger. Typical, non-limiting examples of elastic materials in the context of the present invention are plastics such as polyacrylics; polycarbonates; polyethylene; polyvinyl chloride; polypropylene; thermoplastic elastomers; silicone; high performance plastics e.g. polytetrafluoroethylene plastic, fluorinated ethylene propylene plastic, perfluoroalkoxy plastic, polyetheretherketone plastic; rubbers; and the like. The term 'fluid' refers to a substance (as a liquid) tending to flow or conform to the outline of its container.

The term 'foldable member' refers to a member made of elastic material constructed in such a way that when pressure is applied in the direction of the plane of the foldable member, the member deforms in a zigzag or accordion-type form. Such a form is for instance known from bendable drinking straws as described in e.g. US 5,335,851.

The term 'gelling agent' refers to a compound that assists in changing a mixture into or taking on the form of a gel. Suitable examples of gelling agents are agar, alginic acid and salts thereof, carrageenan, gelatin, hydroxypropylguar and derivatives thereof, locust bean gum (Carob gum), processed eucheuma seaweed and the like.

The term 'indicator' refers to a substance used to show (for example by change of color or fluorescence) the condition of a mixture such as a solution or a gel with respect to the presence of a particular material (for example an acid, a base, oxidizing or reducing agents). For instance, the term 'indicator' may refer to one or more compounds that are known as pH-indicators, but also to one or more compounds that are known as redox-indicators. Also, the term 'indicator' may refer to mixtures of two or more different types of indicators, such as a combination of a pH- and a redox-indicator. In general, when two or more indicators are used, these indicators are co-operating to increase the indicator effect of each of the indicators when taken alone. In the context of the present invention, suitable indicators may be selected from handbooks such as Η.J. Conn's Biological Stains', R. D. Lillie ed., Baltimore, 1969. Preferred indicators are pH-indicators and/or redox indicators. Examples of suitable indicators are Acid Blue 120, Acid Orange 51 , Acid Yellow 38, Alizarin acid, Alizarin Blue, Azure A, Azure B, Basic Blue 3, Brilliant Black, Brilliant Cresyl Blue, Brilliant Crocein MOO, Brilliant Yellow, Bromocresol Green, Bromocresol Purple, Bromophenol Blue, Bromophenol Red, Bromothymol Blue, Chlorocresol Green, Congo Red, m-Cresol Purple, Gallocyanine, Indigo Carmine, Janus Green B, Litmus, Methylene Blue, Nile Blue A, Nitrazol Yellow (also referred to as Nitrazine Yellow), o-Nitrophenol, p-Nitrophenol, 1-10 Phenanthroline, Phenolphthalein, Safranine O, Thionin, Thymol Blue, Toluidine Blue and Xylenol Blue.

The term 'test medium' refers to a solid composition, preferably in the form of a sol or a gel, for instance comprising a gelling agent. However, the person skilled in the art will understand that other types of solid test media may be based on carrier materials such as ceramics, cotton, glass, metal particles, paper, polymers in any shape or form, silicates, sponges, wool and the like. Usually, a test medium contains one or more indicators, however, these compounds may also be added during the test method. The test medium comprises one or more types of test microorganisms as detecting agents. Optionally, the test medium may also contain nutrients, stabilizers, and substances that change the sensitivity to certain antimicrobial compounds in a positive or negative way, and/or viscosity-increasing agents. Examples of substances that change the sensitivity to certain antimicrobial compounds are antifolates like ormethoprim, tetroxoprim and trimethoprim that improve the sensitivity of the test organism towards sulfa compounds or salts of oxalic acid or hydrofluoric acid, which improve the sensitivity towards tetracycline. Examples of compounds that reduce the sensitivity of the test organism towards compounds such as penicillins include compounds such as e.g. cysteine and penicillin-binding protein. Examples of viscosity-increasing agents are ascorbyl methyl- silanol pectinate, carbomer, carboxymethyl cellulose, cetearyl alcohol, cetyl alcohol, cetyl esters, cocamide DEA, emulsifying wax, glucose, hydroxyethyl cellulose, hydroxy- propylmethyl cellulose, lauramide DEA, linoleamide DEA, magnesium aluminum silicate, maltodextrins, PEG-8 distearate, polyacrylamide, polyvinyl alcohol, PVP/hexadecene copolymer, sodium chloride, sodium sulfate, soyamidopropyl betaine, xanthan gum and the like. Alternatively, the optional ingredients of the test medium mentioned above may also be added exogenously. The term 'test microorganism' refers to a microorganism of which the growth is in relation with the presence or absence of the analyte one desires to detect or quantify. For instance, for the detection of antibiotics, the test microorganism preferably is a thermophilic organism such as a Bacillus species, preferably Bacillus stearothermophilus, an Escherichia coli species, or a Streptococcus species, preferably Streptococcus thermophilus. These species may be introduced in the test as units capable of producing colonies, or Colony Forming Units (CFU's). Said CFU's may be spores, vegetative cells or a mixture of both. The concentration of said CFU's is expressed as Colony Forming Units per ml of test medium (CFU. ml^"1) and is usually in the range of 1 x 10⁵ to 1 x 10¹² CFU. ml^"1, preferably 1 x 10⁶ to 1 x 10¹⁰ CFU. ml^"1, more preferably 2 x 10⁶ to 1 x 10⁹ CFU. ml^"1, most preferably 5 x 10⁶ to 1 x 10⁸ CFU. ml^"1, or still more preferably 5 x 10⁶ to 2 x 10⁷CFU. ml^"1.

In a first aspect of the invention, there is provided a device for detecting an analyte in a fluid, an example of which is given in Figure 1 , comprising a first compartment (1 ) having a closed end (1.1 ), an open end (1.2) and a test medium (1.3), and a second compartment (2) having a first open end (2.1 ) connected to open end (1.2) and a second open end (2.2) wherein at least parts of compartments (1 ) and/or (2) are made from elastic material, characterized in that a means (1.4) for decreasing the volume of compartment (1 ) is present. As indicated above, open end (2.1 ) is "connected to" open end (1.2); this refers to the fact that the two open ends can be connected to one another e.g. by means of a separate member or directly, for instance during production by welding the two compartments together using any type of glue or the same material as is used for the production of the compartments. In an embodiment the first and second compartment are inseparable and form one compartment having a closed end (1.1) and an open end (2.2). In another embodiment compartments (1) and (2) are constructed separately, i.e. by injection molding or blow molding, where after compartment (1 ) is partially filled with test medium. When the test medium has sufficiently solidified, compartments (1 ) and (2) are attached to one another e.g. by gluing or, more preferably, by clicking together. Further examples of a device according to the first aspect of the invention are given in Figures 4-6. In the embodiment shown in Figure 4, compartments (1) and (2) are connected (via their open ends (1.2) and (2.1 )) by means of a separate member, i.e. means (1.4). In an embodiment means (1.4) is inserted into compartment (1 ) and compartment (2) fits in means (1.4). Means (1.4) comprises at least one opening (1.5) connecting compartments (1 ) and (2). Through the opening fluid can be transferred between compartment (1 ) and (2). The opening may have a diameter from 0.01-10 mm. When the device is used in the method according to the present invention, fluid will be transferred from compartment (2) to compartment (1 ). When fluid is present in compartment (2), this fluid is not transported into compartment (1) on the basis of gravity alone. For example, under pressure in compartment (1 ) can lead to transferal of fluid from compartment (2) to compartment (1 ). This under pressure can be created by changing the form of means (1.4), e.g. transformation/deformation of means (1.4) or a part thereof, such that the volume of compartment (1 ) is decreased. Means (1.4) or a part thereof can be transformed/deformed by applying external pressure onto the means, e.g. by pushing compartment (2) downwards.

Suitable materials of which the device of the present invention may be constructed are elastic materials as defined above. The complete device may be constructed from one type of elastic material, but in an alternative embodiment different compartments of the device may be constructed from different types of elastic materials. For instance, the second compartment (2) as well as the part of the first compartment wherein the test medium (1.3) is located can be made of polypropylene, while the means (1.4) for decreasing the volume of compartment (1 ) can be made of thermoplastic polymer. In another embodiment means (1.4) can be made of rubber. Such a means is for instance suitable for use in a device as shown in Figures 4-6. Means (1.4) or a part thereof may contain one or more members, e.g. ribs, on the outside and/or on the inside surface. The members can be beneficial in preventing leakage of fluid and/or air. The design of the members on the inside can differ from the design of the members on the outside; however, in an alternative embodiment the design of the members on the inside and outside is equal. The members, e.g. ribs, can point upwards, downwards or can extend perpendicularly from the inside or outside surface of means (1.4).

In yet another alternative embodiment at least parts of compartments (1 ) and/or (2) can be made of non-elastic material, e.g. the part of compartment (1 ) wherein the test medium is present can be made of non-elastic material. The devices of the first aspect of the present invention can all be easily made using standard protocols known to the person skilled in the art. Traditional and well-known processes such as injection molding and blow-molding can be used for the production of all devices of the present invention. Production of these devices can be performed with many types of materials using standard molding equipment, which can be adapted to the required dimensions. Preferably, the device is constructed from a material with sufficient transparency. However, devices according to the invention wherein compartment (2) is made of non- transparent material are also part of the present invention. Sufficient transparency in this respect refers to a transparency that allows for visual and/or mechanical detection of colors and color changes within test medium (1.3) and/or the presence of milk in compartments (1 ) and/or (2). The person skilled in the art is familiar with the types of materials that fulfill both elasticity and transparency requirements. Although injection molding or blow-molding the device from one material in many cases will be the production method of choice for practical and economical reasons, the use of other materials such as glass, metal and other non-elastic materials is by no means excluded, provided that elastic materials are present in means (1.4) and at least parts of compartment (2) and transparent materials are present in at least parts of compartment (1 ) such that the color of test medium (1.3) can be observed. The thickness of the walls of compartments (1 ) and (2) can range from between 0.01 and 10 mm, preferably 0.05 and 5 mm. The device is constructed such that fluid can be sucked in by the creation of a pressure drop. In order to achieve this, at least part of compartment (2) is constructed of elastic material. Preferably, the shape of said material can be changed by applying force. After fluid has been withdrawn into compartment (2), the device is rotated, external pressure is applied onto means (1.4) leading to a decrease of the volume of compartment (1) and the pressure is released again leading to under pressure in compartment (1 ). The difference between the original pressure and the created under pressure in compartment (1) is called delta pressure. The delta pressure can range from 1 to 100,000 Pa, preferably from 5 to 50,000 Pa. As a consequence of the delta pressure, an exact amount of fluid, e.g. 100 μl, is transferred into compartment (1 ) and contacted with test medium (1.3).

Test medium (1.3) comprises a substance with which an analyte can be detected. A device of this type has the advantage that sample withdrawal and measurement can be carried out in one and the same device as the sample analysis. This reduces the amount of operations to be performed by the user, and consequently reduces the occurrence of errors and increases the reliability. Thus, there is provided a device that is partially filled with a test medium such as carrier material and/or a test medium comprising a substance with which an analyte can be detected in a fluid. The person skilled in the art will appreciate that the device may be filled with a wide variety of media that are suitable for detecting various analytes. Such a test medium may contain an indicator or may be an indicator itself. The test medium may be in fluid or solid form and the solid form may be a sol or a gel; the advantage of a solid form is that the test medium stays in place when the device is rotated. Preferably, when the test medium is a sol or a gel, the test medium is introduced in the device in liquid form, i.e. at elevated temperatures (e.g. ranging from 50 to 150⁰C), whereupon the test medium is allowed to solidify, i.e. by cooling to lower temperatures such as ranging from -10 to 50⁰C. Other methods of obtaining this liquid to solid conversion, i.e. photochemical conversions, irradiation and other chemical conversion, may also be employed. The test medium may be any medium suitable for the detection of an analyte in a fluid. It may be a medium comprising or consisting of an indicator.

Preferably, the fluid that is to be withdrawn is a fluid that may or may not contain one or more analytes that are to be detected. Examples of such fluids are beverages, blood, cream, eggs, fruit juices, honey, meat juice, fish juice, poultry juice, milk, milk products, urine and the like. The volume of fluid that is to be withdrawn usually ranges from 0.01-15 ml, preferably from 0.03-1O mI, more preferably from 0.05-5 ml, most preferably from 0.08-3 ml. Examples of analytes that are to be detected are antibiotics, carbohydrates, hormones, metals, microorganisms, nucleic acids, peptides, salts, toxic components and the like.

In a first embodiment, for the detection of antibiotics in fluids, the test medium may comprise a test organism, nutrients for the test organism, a substance that provides a solid state and at least one indicator. Suitable nutrients in this respect are carbon-sources and nitrogen-sources of which many commercially available variants exist. Typical constituents are amino acids, monosaccharides, vitamins and the like. Also oligosaccharides may be present as nutrient. Preferably, the oligosaccharide is partly soluble in aqueous solutions and preferably the oligosaccharide contains one or more glucose units. More preferably, the oligosaccharide is a relatively short-chained oligosaccharide such as a disaccharide, a trisaccharide, a tetrasaccharide or a pentasaccharide. Most preferably, the oligosaccharide is a disaccharide such as cellobiose, gentiobiose, lactose, maltose, sucrose or trehalose. Suitable trisaccharides are maltotriose, melezitose and raffinose. Suitable indicators are those as described above. Particularly useful are indicators that, upon changing from one state to the other, provide a visually detectable signal such as a change in color or fluorescence. The amount of indicator in the test medium is between 0.001 and 50 g/l test medium, preferably between 0.005 and 10 g/l, more preferably between 0.01 and 5 g/l, most preferably between 0.02 and 2 g/l. Preferably, the substance providing for a solid state is a gelling agent and/or a carrier material. The amount of gelling agent in the test medium is between 1 and 200 g/l test medium, preferably between 2 and 100 g/l, more preferably between 3 and 50 g/l, most preferably between 5 and 30 g/l. Preferred gelling agents are agar and gelatin. The amount of test medium present in the device can vary from 0.01 to 10 gram, preferably 0.05 and 5 gram.

In a further embodiment, the diameter of open end (2.2) is such that evaporation of sample and/or medium components is kept to a minimum. In many devices known in the art said evaporation leads to loss in accuracy of the test system. Suitable diameters of open end (2.2) in this respect are from 0.01-2 cm, preferably from 0.05-1 cm, more preferably from 0.1-0.5 cm, most preferably from 0.2-0.3 cm. Although different shapes are conceivable and by no means unsuited for the present invention, the usual shape of the first and second compartments is cylindrical with diameters ranging from 0.2-10 cm, preferably from 0.4-5 cm, more preferably from 0.6-2 cm, most preferably from 0.9-1.4 cm and lengths ranging from 0.5-30 cm, preferably from 1-20 cm, more preferably from 2-15 cm. Normally, the ratio between length and diameter is from 0.1-100, preferably from 1-50, more preferably from 2-20, most preferably from 5-15. At least part of compartment (2) may have a flattened surface and/or other means, e.g. ribs, which provide a good grip of the device. Ideally, unwanted evaporation and/or contamination are prevented by the presence of an optional removable lid at open end (2.2) of second compartment (2). Such a removable lid may be a screw cap, a breakable cap, a tear-off cap, a cap that can be cut off, a film hinge or any other suitable sealing means. When using a device according to the invention having a removable lid, the lid is first removed, for instance by breaking and/or turning and/or tearing or cutting off. Then, the device is placed into the fluid to be analyzed and fluid is withdrawn by applying pressure onto compartment (2). When the fluid withdrawal is complete, the device is turned and pressure is applied onto means (1.4) followed by release of pressure such that eventually part of the fluid in the device comes into contact with test medium (1.3), whereupon the device can be placed in e.g. an incubator. In an embodiment the device comprises means that inform the subject applying pressure onto means (1.4) when sufficient pressure has been applied.

In another embodiment, the diameter of open ends (1.2) and (2.1 ) is such that aqueous and/or fatty fluids cannot pass through the opening based on gravity alone. Preferably, diameters of open ends (1.2) and (2.1 ) in this respect are from 0.005-1.0 cm, more preferably from 0.01-0.8 cm, most preferably from 0.1-0.6 cm. In another embodiment of the first aspect of the invention, said means (1.4) for decreasing the volume of compartment (1 ) comprises at least one foldable member as defined above. The form of such a foldable member is stretched when no pressure is applied, thereby giving a maximum volume to compartment (1 ) and folded, for instance like an accordion, when pressure is applied thereby reducing the volume of compartment (1 ) to its minimal value.

In a further embodiment, the device comprises a drip ring (2.3), which has two distinct advantages. Firstly, the presence of the drip ring prevents any fluid present on the outside of the device following sampling to run down when the device is rotated. Secondly, the drip ring can also function as marker, for instance to indicate the user to which point a device is to be inserted into a fluid. Of course, the device may also contain one or more markers other than a drip ring such as a volume marking on the second compartment which is visible from the outside. When the device is cylindrical, the drip ring is also cylindrical. Likewise, when the device has any other form, the drip ring has the same form as the device.

In yet a further embodiment, the device of the first aspect of the invention may be constructed such that fluid samples can be obtained from solids. For instance, this may be achieved by supplying cutting edges at open end (2.2) by means of which a sample can be cut from solid material such as meat or fish or fruit. Subsequently, a fluid is obtained from the solid material by e.g. applying pressure onto the elastic material of the second compartment of the device.

The devices of the first aspect of the present invention are illustrated in Figures 1-6. The illustrations given in these Figures are not limiting with regard to the shape and size of the device of the present invention. For instance, in Figure 1 compartments (1 ) and (2) are depicted as cylinders. However other forms, such as conical and/or rectangular and/or elliptic forms, are also possible. Likewise, the shape and the ratio of the radii of the compartments in Figures 1-6 are not limited to those depicted. The radius of the first compartment (1 ) may be smaller or larger than that of the second compartment (2). Other deviations are also meant to be included in the invention such as devices having compartments that have a cylindrical shape and end in a conical shape i.e. devices wherein the second compartment becomes a conical shape towards open end (2.2). Devices having a second compartment (2) that is comprised of at least two different sub compartments each having a different shape and/or length and/or radius are also part of the present invention. An example of such a device is depicted in Figures 5 and 6. This device has a second compartment (2) comprising of two distinguishable compartments, a tip end (near open end (2.2)) and a squeeze compartment (near open end (2.1 )). It is of course to be understood that the shape and the ratio of the radii of the compartments in Figures 5 and 6 are not limited to those depicted. In an embodiment the tip end has a length of between 3 and 200 mm, preferably between 5 and 100 mm.

In a second aspect of the invention, there is provided a method for determining the presence or absence of an analyte in a fluid comprising the steps of: (a) inserting the open end (2.2) of the device of the first aspect of the invention into said fluid;

(b) applying external pressure on compartment (2) of said device followed by release of pressure;

(c) withdrawing said device from said fluid; (d) rotating said device such that the angle between a straight line from the centre of open end (2.2) to the centre of closed end (1.1) and level is between 45 and 135 degrees and open end (2.2) is positioned above closed end (1.1 );

(e) applying external pressure on means (1.4) of said device followed by release of pressure such that part of the fluid in said device is contacted with a test medium (1.3) suitable for detecting said analyte. External pressure on means (1.4) can for instance be applied by pushing compartment (2) downwards (into the direction of compartment (1 )).

In an embodiment, for the detection of antibiotics the test medium comprises CFU's of a microorganism and at least one indicator and optionally nutrients for the microorganism. Preferably, the test medium is a sol or gel comprising a gelling agent and/or a carrier material. Advantageously, the method also provides for conditions whereby there is minimal growth of a microorganism prior to the addition of fluid sample. Such conditions comprise an unfavorable temperature and/or an unfavorable pH-value and/or the absence of nutrients essential for growth, provided these conditions do not cause irreversible damage to all CFU's present. After addition of the fluid sample, growth of the microorganism is allowed to take place during a period sufficiently long for the microorganisms to grow in case no antibiotic is present. Growth is encouraged by adding nutrients, optionally before the contacting of said fluid sample, and/or raising the temperature, and/or providing for a pH-value at which the microorganism is able to grow. Alternatively, these conditions may be established prior to contact of the fluid sample with the test medium. Growth of the microorganism is detected by observing the presence or absence of a change of the indicator.

In another embodiment of the second aspect of the present invention, the antibiotic is a β-lactam antibiotic such as a cephalosporin or a penicillin derivative. Examples of such derivatives are amoxicillin, ampicillin, cefadroxil, cefradine, ceftiofur, cephalexin, penicillin G, penicillin V and ticarcillin, but of course many other similar β-lactam derivatives are known and applicable in the method of the present invention.

In yet another embodiment of the second aspect of the invention, the test micro- organism is incubated for a predetermined period, preferably within a time span of 0.5 to 4 hours, more preferably between 0.75 to 3 hours, most preferably between 1.0 to 2.75 hours. Preferably, the microorganism is incubated at a predetermined temperature, preferably the optimal growth temperature of the microorganism. When, for example, thermo stable microorganisms are used, said temperature is preferably between 40 and 7O⁰C, more preferably between 50 and 65⁰C, most preferably between 60 and 64⁰C. Optionally, said reaction could be carried out with the aid of a thermostatic device. Alternatively, the time required for growth of the microorganism is equal to the time that is required for a calibration sample with a known amount of analyte(s) to induce a change in the indicator. The latter change occurs when the concentration of analyte in the sample is below the threshold value.

In a further embodiment of the second aspect of the present invention, nutrients are added as a separate source, e.g. as a tablet, disc or a paper filter. Also other compounds such as the indicator(s), stabilizers and/or antifolates may be added as a separate source, or optionally incorporated in the nutrient medium. The presence or absence of an antibiotic is determined by the presence or absence of a change of the indicator or indicators used. When, for example such a change is a color change, said color change may be observed visually or is determined using an arrangement that generates digital image data or an arrangement that generates analog image data and converts said analog image data into digital image data followed by interpretation of said digital image data by a computer processor, for instance as described in WO 2003/033728.

In a third aspect the invention pertains to a kit comprising one or more devices according to the invention. The devices may be present in an array. The array may have the format of a row containing e.g. 5 or 10 devices connected to each another. The array may also have the format of a matrix containing 5x5 or 10x10 devices connected to each other. The above are only examples of suitable formats; a person skilled in the art knows that other suitable formats can also be used. The kit can be used to carry out the method of the present invention. Optionally, the kit comprises means for sealing the device during incubation; an insert with instructions for use; means for setting the time needed for incubation; nutrients in a medium such as e.g. a tablet, disc or paper filter; indicator(s); stabilizer(s); antifolate(s); a thermostatic device designed in such a fashion that it can hold the devices filled with test medium and optionally coupled to a means for setting the time needed for incubation such that heating and/or cooling is stopped after lapse of a pre-set period; a data carrier loaded with a computer program suitable for instructing a computer to analyze digital data obtained from a sample-reading device; or any combination thereof. The data carrier may be any carrier suitable for storing digital information such as CD-ROM, a diskette, a DVD, a memory stick, a magnetic tape or the like. Advantageously, the data carrier loaded with a computer program also provides for easy access to the latest available computer programs suitable for use in the method of the invention.

Legend to the figures

In Figure 1 a schematic example is given of an embodiment of the device of the present invention. Figure 1A depicts the device in rest, i.e. without any pressure applied.

The device comprises a first compartment (1 ) having a closed end (1.1 ) and an open end (1.2) and a second compartment (2) having two open ends (2.1 and 2.2). Inside first compartment (1), at closed end (1.1 ) a medium comprising a substance with which an analyte can be detected (1.3) is present. Part of the walls of compartment (1 ) have a means (1.4), made of elastic material, for decreasing the volume of compartment (1 ). In

Figure 1 B the device of Figure 1A is depicted, however in the situation when pressure is applied and the volume of compartment (1 ) has decreased through folding of means (1.4).

In Figure 2 a schematic example is given of an embodiment of the device of the present invention wherein, apart from the features shown in Figure 1 , also a drip ring (2.3) is present.

Figure 3 is a schematic representation of an embodiment of the method of the present invention. In Figure 3A compartment (2) is immerged in the fluid (3.1 ) in container (3) to be analyzed and a sample is retrieved through open end (2.2) by manually applying and releasing pressure on compartment (2). In Figure 3B the device is rotated and placed in holder (4), for instance an incubating device, a color reader or both. Pressure is applied in the direction from (2.2) downwards which effectively results in the decrease of the volume of compartment (1 ) through the folding of means (1.4) whereby excess air is released as bubbles passing through the fluid sample. In Figure 3C, the pressure is released and the device resumes its original shape, thereby creating under pressure in compartment (1 ). As a result of this under pressure, an exact part of the fluid sample is transferred from compartment (2) to compartment (1 ), whereas excess sample fluid remains in compartment (2) due to tension forces that prevent transportation through open end (1.2) on the basis of gravity alone. It is to be understood that the device does not to be put into a holder prior to applying pressure in the direction from (2.2). The device can also be put into the holder during or after transfer of the fluid sample from compartment (2) to compartment (1 ). In Figure 3 a device according to Figure 2 has been used to exemplify the steps of the method of the present invention. Of course, also the devices shown in Figure 1 or Figures 4-6 or other devices according to the present invention can be used in the method of the present invention. The pressure in the direction from (2.2) downwards does not necessarily need to be applied by pressing at position (2.2) downwards; the pressure can also be applied by holding compartment (2) between at least two fingers and applying pressure downwards in the direction of compartment (1 ). Any other way of applying pressure effectively resulting in under pressure in compartment (1), e.g. by decreasing the volume of compartment (1 ), can also be used.

In Figure 4 a schematic example is given of a further embodiment of the device of the present invention. Means (1.4) (drawn in black) has been drawn in Figure 4 as if a large part of its outer surface is not in contact with the inner surface of compartment (1 ). This is however only done for clarity reasons, to be able to make a clear difference between means (1.4) and compartment (1) in the drawing. In reality, a large part of the outer surface of means (1.4) is contacted with the inner surface of compartment (1 ). This is necessary in view of the functionality of means (1.4). Under pressure in compartment (1) which is necessary for the device to function can only be created when essentially no air can escape from compartment (1 ). Figure 4A depicts the device in rest, i.e. without any pressure applied onto means (1.4). The device comprises a first compartment (1 ) having a closed end (1.1 ) and an open end (1.2) and a second compartment (2) having two open ends ((2.1 ) and (2.2)). Inside first compartment (1 ), at closed end (1.1 ) a medium comprising a substance with which an analyte can be detected (1.3) is present. Furthermore, the device comprises means (1.4) which is inserted into compartment (1 ). Compartment (1 ) and compartment (2) are connected to one another by means of means (1.4). Means (1.4) in Figure 4 is a separate member made of elastic material that can be used for creating under pressure in compartment (1), e.g. by decreasing the volume of compartment (1). Means (1.4) comprises at least one opening (1.5) through which fluid present in compartment (2) can be transferred to compartment (1). This fluid transferal takes place when pressure is applied in the direction from (2.2) downwards which effectively results in the decrease of the volume of compartment (1 ) through the folding or other change of shape of means (1.4) or a part thereof, whereby excess air is released as bubbles passing through the fluid sample. After release of the pressure, means (1.4) resumes its original shape, thereby creating under pressure in compartment (1). As a result of this under pressure, an exact amount of the fluid sample present in compartment (2) is transferred from compartment (2) to compartment (1 ) through opening (1.5) present in means (1.4), whereas excess sample fluid remains in compartment (2) due to tension forces that prevent transportation through the opening in means (1.4) on the basis of gravity alone. In an embodiment means (1.4) comprises two compartments, one compartment located above opening (1.5) and one compartment located below opening (1.5). Both compartments are connected via opening (1.5). The upper compartment is partly filled with a part of compartment (2). In an embodiment compartment (2) further comprises means (1.6). Through means (1.6) the external pressure applied in the direction from (2.2) downwards is exerted onto means (1.4). In Figure 4B the device of Figure 4A is depicted in the situation when pressure is applied onto means (1.4) and the volume of compartment (1 ) has decreased. Figure 5 shows an example of a three dimensional representation of an embodiment of the device of the present invention wherein, apart from the features shown in Figure 4, also a cap (2.3) is present. This cap can be removed, e.g. by tearing, cutting, pulling or the like, prior to use of the device. In addition, the device comprises means (2.4) for connecting compartments (1) and (2). In the present embodiment part of means (2.4) is present on compartment (1) and another part of means (2.4) is present on compartment (2). In the present embodiment part of means (2.4) extends from compartment (1 ) upwards in the direction of compartment (2). Compartment (2) also comprises part of means (2.4) that fit with part of means (2.4) of compartment (1 ). The parts of the means of compartment (1 ) and (2) can be connected by e.g. clicking. It is to be understood that other means suitable for connecting compartments (1 ) and (2) can also be used. These may be present or be a part of compartment (1) and/or (2). A device containing a single means for connecting the compartments is encompassed in the present invention, but also a device containing more than one means for connection the compartments. If more than one means for connecting compartments (1 ) and (2) are used, these means do not necessarily have to be identical.

Figure 6 shows a further example of a three dimensional representation of an embodiment of the device of the present invention wherein, apart from the features shown in Figure 4, the device comprises a cap (2.5) that differs from cap (2.3) shown in Figure 5. Cap (2.5) should also be removed, e.g. by tearing, cutting, pulling or the like, prior to use of the device. In Figure 6 also means (2.4) for connecting compartments (1 ) and (2) are shown.

Examples Example 1

Preparation of sampler/analyzer for the detection of antibiotics in a fluid A culture of Bacillus stearothermophilus var. calidolactis L. M. D. 74.1 was inoculated on a medium consisting of Bacto nutrient agar, Difco code 0001 (15 g), Bacto agar, Difco code 0140 (5 g), dextrose (0.5 g), MnSO₄-H₂O (30 mg) in distilled water (1000 ml),which was sterilized for 20 minutes at 120⁰C. After inoculation, the medium was incubated at 60⁰C for at least 48 hours until a good sporulation was observed. The spores were then collected, washed with distilled water and stored at 4°C.

The amount of viable spores was detected by testing on a medium consisting of Bacto agar, Difco code 0140 (20 g), Bacto Tryptone, Difco code 0123 (8.5 g), Phytone Peptone, BBL code 11905 (1.5 g), dextrose (5 g) in distilled water (1000 ml), which was sterilized for 20 minutes at 120⁰C. After inoculation, the medium was incubated for 48 hours at 60°C after which the colonies were counted. Distilled water was added to, or water was removed from, the spore suspension until the suspension contained about 10⁸ viable germs per ml. One percent of the above-mentioned spore suspension containing 10⁸ germs per ml was added to an aqueous solution of Bacto agar, Difco code 0140 (12 g/L) and sodium chloride (9 g/L), which was sterilized for 20 minutes at 120°C. The medium was liquefied by heating and then cooled to 60⁰C. Devices of the dimensions of the present invention were obtained by blow molding of plastic and were each filled with 0.5 ml of the medium obtained above and the contents of the devices were allowed to solidify with the devices being held in an upright position. The devices were stored at a temperature of 4°C.

Claims

1. Device for detecting an analyte in a fluid comprising a first compartment (1 ) having a closed end (1.1 ), an open end (1.2) and a test medium (1.3), and a second compartment (2) having a first open end (2.1 ) connected to open end (1.2) and a second open end (2.2) wherein at least parts of compartments (1) and/or (2) are made from elastic material, characterized in that a means (1.4) for decreasing the volume of compartment (1 ) is present.

2. Device according to claim 1 , wherein said means (1.4) is made from elastic material and is inserted into compartment (1).

3. Device according to claim 1 or 2, wherein said means (1.4) comprises at least one foldable member.

4. Device according to any one of the claims 1 to 3, wherein the device, with the exception of test medium (1.3), comprises one elastic material.

5. Device according to any one of the claims 1 to 4 further comprising a drip-ring (2.3).

6. Device according to any one of the claims 1 to 5, wherein the diameter of open ends (1.2) and (2.1 ) is between 0.005 and 1.0 cm.

7. Device according to any one of the claims 1 to 6, wherein said test medium (1.3) comprises a gelling agent, an indicator and a test microorganism.

8. Device according to any one of the claims 1 to 7, wherein said open end (2.2) of compartment (2) is closed with a removable lid.

9. A method for determining the presence or absence of an analyte in a fluid comprising the steps of:

(a) inserting the open end (2.2) of the device of any one of the claims 1 to 8 into said fluid;

(c) withdrawing said device from said fluid;

(d) rotating said device such that the angle between a straight line from the centre of open end (2.2) to the centre of closed end (1.1 ) and level is between 45 and 135 degrees and open end (2.2) is positioned above closed end (1.1);

(e) applying external pressure on means (1.4) of said device followed by release of pressure such that part of the fluid in said device is contacted with a test medium (1.3) suitable for detecting said analyte.

10. Method according to claim 8, wherein said test medium (1.3) comprises an indicator and a test microorganism, further comprising the steps of:

(f) incubating said test microorganism with the fluid under conditions whereby growth of the test microorganism occurs if no analyte is present in the fluid sample; and

(g) detecting any growth or inhibition of growth of the test microorganism as appropriate by means of said indicator,

11. Use of a device according to any one of the claims 1 to 8 for sampling and analyzing a fluid.

12. Kit comprising at least a device according to any one of the claims 1 to 8.