MICROSENSOR

Abstract

The present invention relates to a miniature device for detecting, identifying and/or quantifying microorganisms in a sample. The device comprises a surface for contact with a sample to be analysed, said surface defining a pore and said pore comprising means for reporting the presence of a microorganism. The reporting means may comprise a solid or semi-solid substrate, a metabolic indicator; and a media and/or nutrients that support or encourage microbial growth.

Claims

1. A device for detecting, identifying and/or quantifying microorganisms in a sample, said device comprising a surface for contact with a sample to be analysed, said surface defining a pore, said pore comprising means for reporting the presence of a microorganism characterised in that the reporting means comprises: a solid or semi-solid substrate; a metabolic indicator; and a media and/or nutrients that support or encourage microbial growth but which does not activate the indicator in the absence of a microorganism.

2. The device according to claim 1 wherein the metabolic indicator is a tetrazolium salt.

3. The device according to claim 2 wherein the tetrazolium salt is MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) or MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).

4. The device according to any preceding claim wherein the media and/or nutrients that support or encourage microbial growth is a nutrient broth.

5. The device according to claim 4 wherein the broth does not result cause conversion of a tetrazolium salt into formazan when the broth is incubated with a tetrazolium salt at 40° C. over night.

6. The device according to claim 5 wherein the broth is Mueller Hinton Broth or Wilkins Chalgren Broth

7. The device according to claim 1 wherein the solid or semi-solid substrate is agar or agarose.

8. The device according to claim 7 wherein the substrate is agar and the reporting means comprises about 1% w/v agar or the substrate is agarose and the reporting means comprises about 0.7% w/v agarose.

9. The device according to claim 1 wherein the pore is cylindrical pore and has a diameter of 10 mm.

10. The device according to claim 9 wherein the diameter is less than 5 mm.

11. The device according to claim 1 further comprising a containment layer located between where the sample will be located when in use and the reporting means in the pore.

12. The device according to claim 11 wherein in the containment layer comprises a viscous liquid approximately 100-400 μm thick or deep.

13. The device according to claim 11 wherein in the containment layer comprises carboxymethyl cellulose.

14. The device according to claim 1 further comprising a physical barrier that allows microorganisms to enter the reporting means but retains the components of the reporting means within the pore.

15. The device according to claim 14 wherein the physical barrier is a mesh.

16. The device according to claim 15 wherein the mesh has a mesh pore size of approximately 100 μm.

17. The device according to claim 1 wherein the pore has a volume of less than 250 μl.

18. A contact lens case characterised in that each chamber for retaining a lens comprises a device according to claim 1.

19. A wound dressing characterised in that the dressing comprises at least one device according to claim 1.

20. A method of analysing a sample for the presence of microorganisms, said method comprising the steps of: (a) contacting a device according to claim 1 with a sample to be analysed; and (b) examining the reporting means to determine whether or not microorganisms are present in the sample.

21. A device for detecting, identifying and/or quantifying microorganisms in a sample, said device comprising a surface for contact with a sample to be analysed, said surface defining a pore, said pore comprising means for reporting the presence of a microorganism.

22. A method for detecting, identifying and/or quantifying microorganisms in a sample or on an object, surface or other device, comprising contacting a device according to claim 1 to the sample or the object, surface, or other device; and detecting, identifying and/or quantifying the microorganisms.

Description

DETAILED DESCRIPTION

[0169] The present invention will now be described in detail with reference to the following figures which show:

[0170] FIG. 1 shows a cross-sectional view of a device according to one aspect of this invention.

[0171] FIG. 2 shows the device of FIG. 1 being used to probe a sample for the presence of microorganisms.

[0172] FIG. 3: The MicroSensor device, as manufactured for use within a range of medical devices. The generic structure of the device is shown (A) including the effect of microbial entry leading to the device being triggered (B). This entire unit is designed for separate manufacture, and incorporation into a range of medical devices. The appearance of a prototype of this device is shown in (C). The effect of treatment with 10.sup.6 CFU Pseudomonas aeruginosa followed by overnight incubation is shown in (D).

[0173] FIG. 4: Schematic representations of possible manufacturing processes. (A) continuous processing—hot embossing: (B) continuous processing—hot embossing (including backing): (C) Screen Printing onto laser ablated substrate: (D) Gravure screen printing allowing greater precision in region 4.

[0174] FIG. 5: Typical appearance of the Microsensor Reporting Means after overnight incubation following microbial challenge.

[0175] FIG. 6 A-C: A High Sensitivity Microsensor.

[0176] FIG. 7 A-C: A Low Sensitivity Microsensor

[0177] FIG. 8 is a photograph of 25 devices according to the second aspect of the invention with growth chamber dimensions of 2×2 mm showing triggering of the reporting means in the first two rows.

[0178] FIG. 9 is a photograph of 18 devices according to the second aspect of the invention with growth chamber dimensions of 4×2 mm showing triggering of the reporting means in the first two rows.

[0179] FIG. 10 represents photographs of: (A) a worn wound dressing; (B) a simulated control wound dressing (Smith and Nephew Allevyn Lite non adhesive dressing) moistened with PBS; and (C) 4 devices with growth chambers of 1×1 mm containing growth media and MTT with triggering of the reporting means for the bottom two devices.

[0180] FIG. 11: represents a schematic drawing of: (A) components of a lens case and a device according to the second aspect of the inventon; and (B) an assembled lens case incorporating a device according to the invention.

[0181] FIG. 12A-12C: represents cross-sections of the lens and device depicted in FIG. 12 wherein (12A) is an expanded view of the components of the case and device; (12B) illustrates the device during assembly; and (12C) shows a completed lens case incorporating a device according to the invention.

[0182] FIG. 13: is a photograph of five contact lens cases which have a 4×2 mm Microsensor device at the base of each well according to the second aspect of the invention. The left handside of each case exhibits triggering of the reporting means.

[0183] Turning to FIG. 1, there is shown a device (10) for detecting, identifying and/or quantifying microorganisms in a sample, said device comprising a surface (2: herein after referred to as the sampling surface) for contact with a sample to be analysed (sample not shown here), said surface (2) defining a pore (4), said pore (4) comprising means (6) for reporting the presence of a microorganism.

[0184] In this embodiment, the sampling surface (2) is the upper surface of a substrate (8) of the device (10). The pore (4) defined by the sampling surface (2) comprises a wide opening (4a) at the sampling surface and a narrow opening (4b) below the sampling surface (2: i.e. countersunk therein). As can be seen in FIG. 1, this arrangement forms a dimple or depression in the sampling surface which tapers from the narrow pore opening (4b) to the wide pore opening (4a) to serve as a funnel guiding material from the sampling surface (2) down into the pore (4) and ultimately through the pore shaft (4c). The sides of the funnel formed between wide pore opening (4a) and narrow pore opening (4b) are, in this embodiment coated with functionalising factors (14) which enhance, promote or support microbial colonisation, adherence, binding and/or growth.

[0185] The pore extends from the sampling surface (2) down, through the substrate 8 and terminating in a chamber (12) which contains the means for reporting microorganisms (6)

[0186] FIG. 2 shows the device presented in FIG. 1 in use. Here, a sample 16 which comprises microorganism (18) has been brought into contact with the sampling surface (2) of the device (10). The presence of functionalising factors (14) around the opening (or mouth) of the pore (4), encourages migration of the microorganisms (18) from the sample (16) to the sampling surface (2) where they colonise and grow around the pore openings (4a and 4b). As the microorganisms (18) grow or continue to migrate, they move down the pore (4: through the pore shaft (4c)) toward the reporting means (6) contained within the chamber (12) at the end of the pore (4). Ultimately, the microorganism reaches the reporting means (6) which, in this embodiment, comprises an indicator which undergoes a chemical reaction to produce a pigment when metabolised by a particular microorganism. As such, if the user detects the presence of a dye or pigment in the reporter means (6), he/she can conclude that the sample (16) contained microorganisms (18).

[0187] FIG. 3: A shows the microsensor device prior to use and activation. Where 1) represents the outside of the microsensor surface, 2) represents the graduated opening towards the entry pore, 3) represents the channel leading to the growth chamber, and 4) represents the “inactivated” growth chamber which contains agar, nutrient broth, and inactive dye MTT.

[0188] FIG. 3B shows the device in use, where 1) represents the outside of the microsensor surface, for instance in contact with a wound, 2) represents the graduated pore area with bacteria clearly present, 3) represents the channel with bacteria travelling towards the growth chamber, 4) represents the blue triggered growth chamber with a positive result for the presence of microorganisms/bacteria indicated with, in this case, MTT.

[0189] FIG. 3C represents the visible appearance of an inactivated growth chamber containing agar, nutrient broth and inactive MTT, no bacteria are present.

[0190] FIG. 3D represents the visible appearance of contamination, the blue coloured response as a result of the activation of MTT in response to the presence of microorganisms/bacteria.

[0191] FIG. 4 shows four possible manufacturing processes—each is discussed in more detail below:

[0192] FIG. 4A: Continuous processing using a thin film polymeric substrate (labelled in FIG. 4 as 1a). This substrate may be hot embossed to introduce hemispherical cavities into which an agar-based reagent (FIG. 4—labelled 4) is introduced. The cavity shapes may be varied, but one of skill will appreciate that sharp edges and corners should be avoided—the continuous roll hot embossing process will provide better mould tool release if the features are more “rounded”. The cavities may be filled after the hot embossing of the substrate through either robotic dispensing, or a more simplistic planar filling, using, for example, a squeegee. The final processing stage may comprise the lamination of a backing film (see FIG. 4—labelled 1b). This could be incorporated using full or patterned coating of adhesive. Alternatively, continuous ultrasonic welding could join a backing film of identical polymer to the embossed substrate. The pore opening defined by 1a (in this case an opening of about 1 μm) may be created using laser ablation of the substrate polymer after the hot embossing step.

[0193] b) Continuous processing, similar to that described in a) above, but a hot embossing process has been used to generate a spherical cavity in a backing film (1b).

[0194] c) Screen printing is used to introduce precise volumes of an agar reagent onto a predrilled polymer substrate. This could be a continuous roll process, but it may be necessary to use gravure screen printing to ensure a more precise agar reagent shape and volume. The agar could then be frozen, within a continuous roll process, and a backing material (labelled 1b) added as a liquid film. This film may be solidified by a variety of techniques, including UV-curing etc.

[0195] d) A further application of a screen printing technique, but this time the geometric structure to contain the agar reagent is first formed by gravure screen printing (labelled 1c). Subsequently, the agar reagent is introduced using either robotic dispensing, or planar filling with, for example, a squeegee. Freezing of the agar, would then allow a similar encapsulation, as described in c).

[0196] FIG. 5 shows the results of challenging typical microsensor reporting means, consisting of broth in agar with metabolic indicator, using various levels of broth and MTT, after challenge with S. aureus.

[0197] FIGS. 6A and B show a single microsensor unit (20) as might be found in a highly sensitive microsensor device. In this embodiment, the microsensor unit 20 comprises a pore 22 defined within the sampling surface 24 of a substrate 26. The pore 22 is 500 μm wide and extends 250 μm into the substrate 26. Pore 22 connects to a chamber 28 containing the reporting means 30 which, in this embodiment, comprises 0.3% (W/v) agar, 1.5× Mueller-Hinton broth and MTT at 100 μg/ml. The chamber 28 is cylindrical and about 1 mm in diameter and extends about 2 mm into the substrate 24 (in other words the chamber is about 2 mm deep). Between the pore 22 and the chamber 28 containing the reporting means 30, is provided a barrier or containment layer (32). This layer 32 prevents desiccation and egress of components of the reporting means 30 from the chamber 28 and is about 50 μm deep.

[0198] One of skill will appreciate that since the sampling surface 24 of the substrate 26 defines a wide/short pore 22, the sensitivity of the device is high as low numbers of microorganisms can readily and quickly find their way into the chamber 28 containing the reporting means 30 to activate the device.

[0199] FIG. 6C shows an array of microsensor units 20 in a microsensor device 40. In this embodiment, the device 40 comprises 8×8 microsensor units. In this embodiment, the distance between the central point (i.e. the centre of each pore 22 defined by the surface 24 of the substrate 26) of each microsensor unit 20 from the central point of a neighbouring microsensor unit 20, is about 2.5 mm.

[0200] It should be appreciated that the density of microsensor units 20 in the device greatly increases the ability of the device 40 to detect the presence of microorganisms in a sample.

[0201] FIGS. 7A and B show a single microsensor unit (50) as might be found in a low sensitivity microsensor device. In this embodiment, the microsensor unit 50 comprises a pore 52 defined within the sampling surface 54 of a substrate 56. The pore 52 is 250 μm wide and extends 500 μm into the substrate 56. Pore 52 connects to a chamber 58 containing the reporting means 60 which, in this embodiment, comprises 0.3% (W/v) agar, 1.5× Mueller-Hinton broth and MTT at 100 μg/ml. The chamber 58 is cylindrical and about 1 mm in diameter and extends about 2 mm into the substrate 54 (in other words the chamber is about 2 mm deep). Between the pore 52 and the chamber 58 containing the reporting means 60, is provided a barrier or containment layer (62). This layer 62 prevents desiccation and egress of components of the reporting means 60 from the chamber 58 and is about 50 μm deep.

[0202] One of skill will appreciate that since the sampling surface 54 of the substrate 56 defines a narrow/long pore 52, the sensitivity of the device is low as it is more difficult for microorganisms to find their way into the chamber 58 containing the reporting means 60 to activate the device. In this way, only samples with high levels of microbial contamination may activate the device.

[0203] FIG. 7C shows an array of microsensor units 50 in a microsensor device 70. In this embodiment, the device 70 comprises 4×4 microsensor units 50. In this embodiment, the distance between the central point (i.e. the centre of each pore 52 defined by the surface 54 of the substrate 56) of each microsensor unit 50 from the central point of a neighbouring microsensor unit 50, is about 2.5 mm.

[0204] It should be appreciated that the low density of microsensor units 50 in the device 70 decreases the sensitivity.

[0205] FIG. 8 is a photograph of 5×5 prototype microsensor units 80. Each unit had 2×2 mm cylindrical pores and are discussed in more detail in Example 2.

[0206] FIG. 9 is a photograph of 3×6 prototype microsensor units 90. Each unit 4×2 mm cylindrical pores and are discussed in more detail in Example 2.

[0207] FIG. 10 is a photograph of 4×4 prototype microsensor units 100 that may be suitable for incorporating into wound dressings. Each unit had pores with 1×1 mm openings and are discussed in more detail in Example 3.

[0208] FIGS. 11 A and B show a contact lens case (110) which has been adapted to incorporate microsensor units (120) in holes (111) formed in the left and right chambers of the case. Microsensor units (120) for joining to the case (110) comprise a sensor housing (121) made out of polypropylene in which a pore (122) and recess (123) are machined in the surface of the housing. The pore (122) is adapted to receive approximately 25 μl in volume and the recess (123) defines an area above the pore (122) which is adapted to fit a mesh (124, a barrier layer according to the invention). The mesh may typically be a PMMA mesh disk with 100 μm mesh pores that, in use, allows bacteria to enter the pore but prevent the reporting means being detached from the pore. The case (110) and device (120) are assembled using a suitable adhesive (125) that does not obstruct communication between the pore (122) and the chambers of the case (110). The adhesive (125) may be an acrylic adhesive such as 3M double sided acrylic adhesive 468MP 200MP.

[0209] FIG. 12A-12C represents cross-sections of the contact lens case (110) and a microsensor unit (120) as illustrated in FIG. 11. FIG. 12A as an expanded illustration of the contact lens case (110), the sensor housing (121), mesh (124) and adhesive (125). The sensor housing (121) as illustrated has a pore (122) which has been filed with first a reporting means (126) and then a containment layer (127) above it. The reporting means is typically agar made up with a nutrient broth and further comprising a tetrazolium salt as an indicator. The containment layer (127) may comprise a viscous liquid (e.g 5% carboxymethyl cellulose in PBS) which allows bacteria to enter the microsensor unit (120) while at the same time maintaining the viability of the reporting means before use (e.g. when the assembled case is in storage). FIG. 12B illustrates the device during assembly wherein the mesh (124) is placed in the recess (123) of the sensor housing (121). FIG. 12C illustrates an assembled case (110) and microsensor (120). It should be noted that the adhesive (125) fixes the case (110) and microsensor (120) together and that mesh (124) and recess for retaining it (123) are dimensioned such that the adhesive (125) also holds the mesh (124) in place.

Example 1

[0210] The inventor realised that there were no commercially available products that were small and simple for detecting microbial contamination of samples or objects. Initial experiments were therefore conducted to evaluate whether or not a detectable signal of microbial contamination could be generated in a simple miniaturised system.

1.1 Materials & Methods

[0211] 1.1.1. Initial experiments evaluated whether a disk of agar containing MTT which was encased in a polymer could develop a visible blue signal when exposed to a bacteria.

[0212] Drops of tryptone soya broth containing 0.5% (w/v) agar were placed on a petri dish and allowed to set. Silicone was poured over the droplets and set at 80° C. for 1 hour. Once set, disks were cut out using a cork borer. 2 μL 5 mg/mL MTT was added to the agar and allowed to soak in. The silicone agar disks impregnated with MTT were placed in a petri dish. Pseudomonas aeruginosa from an overnight culture was re-suspended and diluted to ˜10.sup.9 cfu/mL in PBS. 1 μL of suspension (containing ˜10.sup.6 cfu Pseudomona aeruginosa) was exposed to the disks and incubated at 37° C. overnight in a humidity chamber. Controls were exposed to 1 μL sterile PBS.

1.1.2. Following the work conducted in 1.1.1 the inventors decided to evaluate whether or not a positive signal could be achieved in varying concentrations of agar.

[0213] Mueller-Hinton broth was prepared containing 1%, 0.7%, 0.5% and 0.3% (w/v) agar, added to the wells of duplicate 24-well microtitre plates and allowed to set in air. Overnight suspensions of microbial cultures (Staphylococcus aureus, Staphylococcus epidermidis, EMRSA, Pseudomonas aeruginosa, Escherichia coli, Bacillus cereus, Enterococcus faecalis, Klebsiella pneumoniae, Serratia marcescens and Candida albicans) were diluted to 10.sup.7 cfu/mL and 500 μL added to the agars. Plates were incubated overnight at 37° C. and room temperature respectively.

1.1.3. Further work examined whether or not variations in the concentration of broth and indicator (MTT) would affect signal generation.

[0214] Mueller-Hinton broth at 1×, 1.5×, 2× and 2.5× normal strength, was prepared with 0.3% (w/v) agar. MTT solubilised in distilled water was sterilised and added to the sloppy agar to give final concentrations ranging 20 to 200 μg/mL in 20 μg/mL increments. The range of agars were added in a chequerboard style, with broth concentration increasing with each row, and MTT increasing with each column, to the wells of a 96-well microtitre plate and allowed to set in air. Overnight suspensions of microbial cultures (Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans) were diluted to 10.sup.7 cfu/mL and 50 μL, added to the agars. Plates were incubated overnight at room temperature.

1.2 Results

[0215] 1.2.1 FIG. 3D represents the typical appearance of triggered disks made according to method 1.1.1 after overnight incubation following microbial challenge with Pseudomonas aeruginosa. FIG. 3C represents the typical appearance of silicone disks containing agar and MTT, but not exposed to Pseudomonas aeruginosa, and incubated in the same petri dish as the exposed sensors remained colourless.
1.2.2 Each of the agar concentrations tested as outlined in 1.1.2 sustained micro-organisms overnight and resulted in the triggering of MTT such there was a colour change from translucent yellow to strong blue (observed for all microorganisms grown at both 37° C. or room temperature) (data not shown)
1.2.3 FIG. 5 shows the data generated following the protocol outlined at 1.1.3 above for Staphylococcus aureus. Agar colour change from translucent yellow to blue was observed for all agar/MTT combinations, with intensity increasing with each broth and MTT concentration increase. Similar data were obtained for the other organisms.

1.3 Discussion

[0216] These data made the inventors realise that it would be possible to generate simple “Microsensor” devices that are capable of reliably triggering following challenge with a microbial suspension. Triggering of the indicator resulted in an unequivocal dark purple visual signal appearing overnight after challenge, without any need for operator intervention. Such a signal can be easily recognised by an observer, who is made aware that the sensor has come into contact with an abnormally high level of microbes.

Example 2

[0217] Having established that reporting means (Example 1) could be triggered by micro-organisms of interest, the inventors proceeded to make prototype devices according to the invention and tested whether or not such devices could be selectively and sensitively triggered by micro-organisms of interest.

2.1 Materials & Methods

[0218] 2.1.1 Prototype devices
2.1.1.1 The prototype devices illustrated in FIG. 8 were formed by the following steps: [0219] (a) A device housing was manufactured by injection moulding using crystal polystyrene. The mould defined the housing, surface of the device and a 2×2 mm cylindrical pore (diameter×depth). The overall dimensions of the devices were 10 mm×10 mm×3 mm. The device was then sterilised. [0220] (b) making a solution of 0.7% agarose in 1× Mueller Hinton Broth. This was then autoclaved. After cooling (to 40° C.), a sterile filtered stock solution of MTT is added to a concentration of 500 μg/ml to form the reporting means. [0221] (c) 6 μl of the reporting means was then added to the pore of the device using a Hamilton Syringe and allowed to solidify for an hour.
2.1.1.2 The prototype devices illustrated in FIG. 9 were formed by the following steps: [0222] (a) A device housing was manufactured by injection moulding using crystal polystyrene. The mould defined the housing, surface of the device and a 4×2 mm cylindrical pore (diameter×depth). The overall dimension of the device were 10 mm×10 mm×3 mm. The device was then sterilised. [0223] (b) making up Wilkins Chalgren agar (1% agar). This was then autoclaved. After cooling (to 40° C.), a sterile filtered stock solution of MTT is added to a concentration of 500 μg/ml to form the reporting means. [0224] (c) 25 μl of the reporting means was then added to the pore of the device using a Hamilton Syringe and allowed to solidify for an hour.
2.1.2 Protocols for testing triggering
2.1.2.1: Testing of Devices with 2×2 mm cylindrical pores.

[0225] Stock suspensions of Pseudomonas aeruginosa ATCC9027 were made as 10.sup.9, 10.sup.8, 10.sup.7, 10.sup.6 and 10.sup.5 cfu/ml stocks. 1 μl of each stock was inoculated onto the top of the pore of a device to give 10.sup.6, 10.sup.5, 10.sup.4, 10.sup.3 or 10.sup.2 cells on each device.

[0226] The devices were then placed in an incubator and left overnight at 30° C. The colour change was observed the next day.

2.1.2.1: Testing of Devices with 4×2 mm cylindrical pores.

[0227] Stock suspensions with an optical density (570 nm) of one were made up for Staphylococcus aureus ATCC 6538 (10.sup.8 cfu/mL); Pseudomonas aeruginosa ATCC9027 (10.sup.8 cfu/mL); Candida albicans ATCC 10231 (10.sup.7 cfu/mL) Serratia marcesens ATCC 13880 (10.sup.8 cfu/mL) and Fusarium solani ATCC 36031.

[0228] 1 ml of each 1OD stock was inoculated onto the top of device retained in a 24 well plate. The plate was then left overnight at room temperature and the colour change observed the next day.

2.2 Results

[0229] 2.2.1 Sensing of a range of micro-organisms

[0230] FIG. 8 is a photograph of 25 devices (prepared according to 2.1.1.1) with growth chamber dimensions of 2×2 mm which had been inoculated with Pseudomonas aeruginosa ATCC9027 or control solution (no micro-organism) according to 2.1.2.1. After a 1 μL inoculation of each device, the top row of devices shows reproducible triggering with a dose of 10.sup.6 cfu (top row) or 10.sup.5 cfu (second row) of Pseudomonas aeruginosa. The third and fourth rows show no triggering at 10.sup.4 and 10.sup.3 cfu, with the fifth row representing a phosphate buffered saline control. This data confirms the 2×2 mm devices containing growth media and MTT are reproducible with respect to both triggering and cfu sensitivity. Similar results were obtained with Staphylococcus aureus, Candida albicans, Serratia marcesens, and Fusarium solani (data not shown).

2.2.2 Sensing of a micro-organisms associated with ocular infection

[0231] FIG. 9 is a photograph of 18 devices with pore dimensions of 4×2 mm (prepared according to 2.1.1.2) which had been inoculated with micro-organisms or control solution according to 2.1.2.2). The photograph shows triggering of the reporting means (in triplicate) with the five ISO standard contact lens organisms. From left to right the first column represents a phosphate buffered saline control, the second Staphylococcus aureus ATCC 6538 10.sup.8 cfu/mL, third Pseudomonas aeruginosa ATCC 9027 10.sup.8 cfu/mL, fourth Candida albicans ATCC 10231 10.sup.7 cfu/mL, fifth Serratia marcesens ATCC 13880 10.sup.8 cfu/mL, sixth Fusarium solani ATCC 36031 (1 OD) overnight culture re-suspended in PBS. This data demonstrates that the 4×2 mm devices are capable of triggering reproducibly in the presence of typical organisms associated with ocular infection.

2.3 Discussion

[0232] These data illustrated that prototype devices according to the invention were able to detect micro-organisms in a reproducible way and in particular illustrated that devices according to the invention may be useful in the management of ocular infection (e.g. devices used according to the third aspect of the invention).

Example 3

[0233] The inventors proceeded to test devices according to the invention in the context of a real situation by evaluating the usefulness of the devices for detecting microbial contamination of a wound dressing.

3.1 Materials & Methods

3.1.1 Moulding of Device Housing

[0234] Device housings were manufactured by a third party from PMMA with SU8 epoxy by moulding with 1×1 mm pores (a volume of approximately 1.6 μl). A silicon coating (PDMS) was applied to the surface to make it easier to identify the surface of the device containing the opening to the pore.

3.1.2 Filling the Device Housing

[0235] (a) A solution of 0.7% agarose in 1× Mueller Hinton Broth was made. This was then autoclaved. After cooling (to 40° C.), a sterile filtered stock solution of MTT was added to a concentration of 500 μg/ml to form the reporting means. [0236] (b) 1.4 μl of the reporting means was then added to the pore of the device using a Hamilton Syringe and allowed to solidify for an hour. 0.2 μl of 5% carboxycellulose (in PBS) was placed on top of the reporting means as a containment layer

3.1.3 Wound Dressings

[0237] A wound dressing (Smith and Nephew Allevyn Lite non adhesive dressing) worn by a patient with a microbial infection was obtained with the consent of the patient. FIG. 10A is a photograph of the worn wound dressing.

[0238] A clean/unused dressing (Smith and Nephew Allevyn Lite non adhesive dressing) moistened with sterile PBS was used as a control. FIG. 10B is a photograph of the simulated control wound dressing.

3.1.4 Protocols for Testing Contamination of a Wound Dressing

[0239] Devices were placed in a petri dish and either a worn dressing or unused dressing pressed on top. The dish and dressing were left over night at 30° C. and any colour change observed the next day.

3.2 Results

[0240] FIG. 10C is a photograph of four devices according to the invention. The top two devices were placed into contact with the control dressing and show no triggering of the indicator whereas the lower two devices were placed in contact with the worn dressing and demonstrated a positive signal in both cases. This confirms that devices according to the invention are capable of detecting infection within a clinical environment.

3.3 Discussion

[0241] These data illustrate that devices according to the invention are capable of detecting infection within a wound environment. A skilled person will appreciate that the devices may be adapted such that they may be incorporated as an array of microsensors within the fabric of a wound dressing. In use, a dressing may be lifted from a wound and inspected. Activation of devices (dark dots within the pores of the devices) will indicate that an infection is present in the wound area and such knowledge may be used to direct future actions. For instance a decision may be taken to at least change the dressing. A clinician may also wish to consider the extent to which the wound is infected and may wish to initiate a course of antibiotics.

Example 4

[0242] The data presented at 2.2.2 inspired the inventors to develop devices according to the invention that may be incorporated into contact lens cases. The inventors realised that devices according to the invention may be used to inform a user that the case and/or the solution within it and/or lenses per se placed in the case have been contaminated by micro-organisms. The user may then decide whether or not to discard the solution; discard or clean the lenses; and/or to discard the lens case as appropriate and thereby reduce the risk of developing an eye infection by introducing a contact lens into an eye which has come from a contaminated lens.

[0243] Contact lens cases according to the third aspect of the invention were made by the following procedures:

4.1. Manufacture of a Housing for the Device

[0244] A device housing was manufactured by injection moulding using either white or crystal polystyrene. The inventors found that a white housing allowed a user to better observe a colour change from inside the chamber of a contact lens case whereas crystal polystyrene allowed a colour change for observing from outside (from below) the case.

[0245] A 4×2 mm cylindrical pore (diameter×depth) with a volume of approximately 25 μl was provided in the moulding. A recess (for receiving a mesh) was provided in the surface of the housing containing the opening to the pore. The device housing was then sterilised.

4.2 Preparation of a Reporting Means

[0246] Wilkins Chalgren agar (1% agar) was made up and then autoclaved. After cooling (to 40° C.), a sterile filtered stock solution of MTT was added to a concentration of 500 μg/ml to form the reporting means.

4.3 Charging the Pore of the Device

[0247] 20 μl of the reporting means was then added to the pore of the device using a Hamilton Syringe and allowed to solidify for an hour. 5 μl of 5% carboxycellulose (in PBS) was then placed on top of the reporting means as a containment layer in the top of the pore.

4.4. Fitting a Physical Barrier

[0248] A PMMA mesh disk with 100 μm mesh was then placed in the recess provided with in the moulded housing.

4.5 Assembly with a Contact Lens Case

[0249] Contact lens cases were obtained and 4 mm diameter holes drilled in the bottom of the case chamber which receive lens solution and a lens when in use. If the type of case required it, the base of the contact lens case was machined and flattened to improve adherence of the device.

[0250] 3M double sided acrylic adhesive 468MP 200MP was the affixed to the underside of each lens case chamber and the lens case careful placed on top of the device according to the inventions (such that the pores and holes align) and allowed to adhere thereto.

[0251] The devices are further illustrated in FIGS. 11 and 12 and further described in the specific description above.

Example 5

[0252] The inventors proceeded to test the devices of Example 5 to evaluate whether or not the reporting means is triggered by inoculation of the chambers of the contact lens case with organisms associated with ocular infection.

5.1 Materials & Methods

5.1.1 Devices

[0253] Devices were made as described in Example 4.

5.1.2 Protocols for Testing Contact Lens Cases Incorporating Microsensor Devices

[0254] Stock suspensions with an optical density (570 nm) of one were made up for Staphylococcus aureus ATCC 6538 (10.sup.8 cfu/mL); Pseudomonas aeruginosa ATCC9027 (10.sup.8 cfu/mL); Candida albicans ATCC 10231 (10.sup.7 cfu/mL) Serratia marcesens ATCC 13880 (10.sup.8 cfu/mL) and Fusarium solani ATCC 36031.

[0255] 1 ml of each 1OD stock was inoculated into the lens holding chambers of the contact lens case. The case was then left overnight at room temperature and the colour change observed the next day.

5.2 Results

[0256] FIG. 14 is a photograph of five contact lens cases which each have a device according to the invention at the base of each lens chamber/well.

[0257] The right hand chamber of each of the lens cases was exposed to a control phosphate buffered saline solution. The devices were not triggered and retained their negative yellow colour. The left hand side of each lens case was exposed to contact lens ISO organism strains from top to bottom S. aureus ATCC 6538 10.sup.8 cfu/mL, P. aeruginosa, ATCC 9027 10.sup.8 cfu/mL, C. albicans ATCC 10231 10.sup.7 cfu/mL, S. marcesens ATCC 13880 10.sup.8 cfu/mL, F. solani ATCC 36031 overnight culture and then re-suspended in PBS. Each device demonstrates a clearly visible positive result demonstrating the suitable application of the device in detecting contact lens related infections.

5.3 Discussion

[0258] These data clearly show that devices according to the invention are particularly useful for detecting microbial contamination of contact lens cases. The devices described in Example 4, and variants thereof, may be cheaply and easily mass produced and are of great utility to people who wear contact lenses and wish to minimise the risk of developing an eye infection.