JP2018533002A - Biosensor and method for detecting microorganisms - Google Patents

Abstract

Biosensors for the immunological detection of microorganisms (50) are planned. The biosensor is finished in the form of a lateral flow test strip (10), which has a microorganism-specific antibody (40). When the antibody (40) and the microorganism (50) to be detected form a complex, the mobility characteristic of the antibody (40) changes, and this change in mobility characteristic is used for detection of the microorganism (50). .

Description

  The present invention relates to a biosensor in the form of a lateral flow test strip and a method for detecting microorganisms.

Prior art Significant microbial contamination loads can occur in various areas of the human living environment. Mold damage, in particular, is not uncommon and can cause serious health problems. Toxic substances secreted from filamentous fungi, particularly filamentous fungi spores and filamentous fungi, should not be overlooked as pathogens for infections and chronic diseases such as inflammatory lung diseases. Furthermore, filamentous fungi and spores of filamentous fungi are potential allergens that can cause allergic symptoms. Damage from filamentous fungi or generally microbial contamination loads can occur especially in various home appliances such as washing machines and coffee makers. Recent washing machines have complicated programs that do not use high temperatures for the purpose of reducing energy. The presence of wet areas for long periods of time, low temperatures during washing operations, and the presence of food residue leave the microorganisms in good growth conditions. Microorganism damage can occur at the laundry detergent inlet. Modern coffee makers, especially fully automatic coffee makers, are often exposed to microbial contamination. This is because the structure is complicated and there are various ranges that are more difficult to clean and reach than the conventional filter coffee maker. Various wet locations (eg, water tanks, tubes, grounds, mills) in modern coffee makers provide very good growth conditions for bacteria and fungi in some cases.

  For coffee makers and washing machines, several cleaning measures have already been proposed, but at that time, a specific cleaning program or cleaning program is performed using a special cleaning agent (tablet or cleaning solution). It is desirable to reduce the microbial contamination load. In practice, however, these cleaning measures are probably not taken regularly and you will not be aware of the occurrence of a microbial contamination load, which can cause health problems for the user. .

  From the description of International Publication No. 2012/0889417 (WO 2012/0889417 A1), a manual measuring instrument for detecting mold damage that has not been clarified is known. Thereby, for example, damage caused by mold in an indoor space such as a wall can be detected. This equipment comprises in particular a collection unit for taking a room sample and an immunological test unit.

  German Offenlegungsschrift 19604195 (DE 196 04 195 A1) discloses an analytical method in which an antibody or antigen is sucked from room air and then detected by an immune reaction.

DISCLOSURE OF THE INVENTION Advantages of the Invention The present invention relates to a biosensor for immunological detection of microorganisms, finished according to the present invention as a lateral flow test strip with microorganism-specific antibodies. When this antibody and the microorganism to be detected form a complex, the mobility characteristics of this antibody change. According to the present invention, by utilizing this change in mobility characteristics, microorganisms can be detected using this lateral flow test strip in a particularly simple and easy-to-use manner.

  Preferred embodiments of the biosensor are the subject matter of the dependent claims.

  The biosensor based on immunological detection according to the present invention is very sensitive and can detect microorganisms that may occur at relatively low concentrations. Furthermore, since a highly specific antibody is used, the selectivity is extremely high. For this reason, the biosensor according to the present invention can be used to detect, in some cases, the damage caused by microorganisms that have just occurred, at an early stage, so that significant microorganisms that affect health can be detected. Appropriate measures can be taken before the contamination load occurs, for example, cleaning of household appliances with a microbial contamination load.

  According to the invention, the biosensor is finished in the form of a lateral flow test strip. A lateral flow test is to be understood as a detection method based on a combination of thin layer chromatography and immunostaining. In this case, the sample moves over the length of the test strip based on capillary force (thin layer chromatography), but the sample may also be accompanied by bound antibody. According to the present invention, when the antibody and the microorganism to be detected form a complex, the mobility characteristic of the antibody changes, and thus the mobility characteristic depends on the presence of the microorganism to be detected. Can be detected.

In one particularly preferred embodiment of the lateral flow test strip, at least one detection reagent is bound to the microorganism-specific antibody. This detection reagent is preferably bound directly or directly to the microorganism-specific antibody. These detection reagents can be immobilized (bound) via an amino acid residue, for example, by a covalent bond. For example, coupling via COOH / NH ₂ is possible. In addition, other reactive groups of the antibody, such as cysteine / SH groups, are suitable for chemical conjugation with detection reagents using a linker system.

  Preferably, the detection reagent is a metal particle or a dye molecule. Thus, for example, an optical reading system capable of detecting the presence or absence of a microbial contamination load based on a color reaction that can be read with the naked eye can be realized in a very easy-to-use manner. In particular, quantitative detection can generally be confirmed only inaccurately by visual observation, or cannot be confirmed by visual observation at all. Accordingly, other embodiments use optical instruments for optical reading or interpretation, such as cameras, which can also be used for quantitative or semi-quantitative evaluation, especially based on measurement of color gradation or luminance gradation. You can also This interpretation can be performed with the aid of software, and at that time, for example, comparison with a registered reference value (calibration software) can be performed. In a preferred embodiment, a mobile phone camera, for example, can be used as the optical device.

  In a preferred embodiment of the biosensor, a biosensor for detecting filamentous fungi and / or filamentous fungal spores and / or bacteria is contemplated. The aforementioned microorganisms are most relevant to the microbial contamination load that can occur in home appliances. Damage caused by bacteria or filamentous fungi, or spores of filamentous fungi, especially in places where the appliance has been wet for a long time, such as water tanks or tubes, or where wet material has accumulated for a relatively long time, for example extraction Occurs in places like a waste container. By selecting a microbial specific antibody corresponding to the biosensor according to the invention, the biosensor can be adapted for the detection of a given microorganism. At this time, it is preferable to examine in advance microorganisms that may be the main cause of the load, depending on the device and use case. These microorganisms can vary from place to place, for example for coffee makers (for example, bacteria in tanks and molds in mills). Many suitable antibodies are commercially available. Monoclonal antibodies may be preferred over polyclonal antibodies. This is because monoclonal antibodies are generally more selective. On the other hand, polyclonal antibodies can also be advantageously used particularly from the viewpoint of cost.

  Particularly preferably, the microorganism-specific antibody corresponds to a surface molecule of the microorganism to be detected. In so doing, the antibody may preferably correspond to an epitope on the surface of the microorganism. This has the advantage that no further processing of the sample is required, for example cell destruction. Since the microorganism-specific antibody can directly bind to the microorganism to be detected, immunological detection can be performed very easily.

  In a particularly preferred embodiment of the biosensor, antibodies corresponding to β-glucan of filamentous fungal spores are used. However, other antigens resident on the surface are also suitable as epitopes for antibodies that can be used according to the invention. Accordingly, the biosensor can be adapted for the detection of a given microorganism by selecting the appropriate antibody and the appropriate detection system accordingly. Thus, for example, a test strip adapted to detect filamentous fungi and a test strip adapted to detect predetermined bacteria can be provided. Furthermore, different test strips can be used in parallel or in sequence for the detection of a given microorganism. Furthermore, it may be finished so that various microorganisms can be detected in parallel by one test strip itself.

  In one particularly preferred embodiment of the biosensor, the lateral flow test strip is designed as a disposable article. The test strip can be discarded after performing a microbial contamination load test using a biosensor, and the microorganisms collected and analyzed on the test strip can be directly disposed of as general waste along with the test strip. . In so doing, the microorganisms stored on the test strip are usually not harmful substances. If the lateral flow test strip is finished as a disposable article, such articles have the additional special advantage that they can be manufactured at a very low overall cost.

  In a preferred embodiment of the biosensor, the lateral flow test strip includes a sampling pad and / or capillary and / or at least one detection region. The sampling pad is utilized not only for sampling but also for binding to assay components (antibodies) and is preferably adapted with respect to pore size and binding properties to the sample. This sampling pad may be formed, for example, from nitrocellulose and may be realized, for example, in the form of a membrane. Furthermore, for example, cellulose or glass fiber is suitable. The test strip itself can be based, for example, on a plastic strip as a support. The expression “capillary” means the range of the test strip that can pass through based on the capillary force of the liquid. In general, liquid permeable materials such as nitrocellulose, polyvinylidene fluoride, nylon (modified chargeability) or polyethersulfone are suitable for this. In addition, the lateral flow test strip preferably includes a verification area. Therefore, the user can examine whether the test result is positive or negative by directly comparing the confirmation region and the detection region, for example. The microorganism-specific antibody may be applied to the test strip, for example in a dried form, or may be added from a separate solution. The capillaries are finished so that they can be separated by thin layer chromatography after a complex of antibody and optionally present microorganisms is formed. In addition, the test strip can include one or more buffers, for example, an uptake buffer and / or a detection buffer and / or a running buffer. Alternatively, a buffer cartridge may be incorporated in the lateral flow test strip, and a necessary buffer may be appropriately discharged from the cartridge. In this test strip embodiment, the sample is applied to the sampling pad in liquid or solid form. Then, a necessary buffer, for example, a running buffer is released from the buffer cartridge, and this buffer is placed in the range of the sampling pad, for example, so that the sample can be transported by capillary force. When placing a sample in liquid form (eg, a water sample taken from a water supply tank), it may be sufficient to separate the sample by thin layer chromatography using only the liquid from this sample. In this case, a separate running buffer is not necessary.

  When examining samples in dry form (eg coffee residue taken from the mill), it is necessary to add the buffer manually or automatically. For example, the buffer previously stored in the buffer cartridge can be released manually or automatically and applied to the sampling pad directly or through a flow path, for example, by capillary force. The buffer cartridge (eg, a plastic container) may be finished, for example, so that the buffer can be released by breaking.

  Depending on the antibody and detection system, it may be necessary to completely or partially degrade the cell (disrupt the microorganism) in order to analyze the microorganism. In this case, a cytolytic reagent such as a surfactant is preferably placed on the sampling pad through a buffer solution. By doing so, depending on the case, if the sample is liquid, the buffer can be placed.

  By separation by thin layer chromatography, the antibody reaches the detection and / or confirmation region of the lateral flow test strip, possibly in the form of a complex with the microorganism to be detected. It is preferable that the user can visually recognize the test result by color reaction or color change. For this purpose, a colored reading system is particularly preferred, whereby no further auxiliary means for reading the test strip are required. In another embodiment, an optical device such as a mobile phone camera can be used for reading.

  In a particularly preferred embodiment, the biosensor according to the present invention is adapted or scheduled to immunologically detect microorganisms in household appliances, for example detecting microorganisms in washing machines and coffee makers. It is adapted or scheduled to do so. In particular, in the case of devices that generally have locations that have been wet for a long period of time, harmful microbial contamination loads can occur very quickly. When the biosensor according to the present invention is used, the user can quickly and easily detect such a microbial contamination load even if he / she does not have special knowledge or ability about it. Therefore, the biosensor according to the present invention allows the user himself / herself to check a place where there is a possibility of damage in a washing machine or a coffee maker. At that time, the user can obtain a qualitative, semi-quantitative or optionally quantitative result after the test is performed, and if the test result is positive, an appropriate cleaning measure can be taken as appropriate.

  In so doing, the form and embodiment of the biosensor or lateral flow test strip may be adapted to a scheduled sampling location within the home appliance. In the simplest case, the lateral flow test strip may be finished in a normal stick shape. However, in particular, it may be planned that the test strip is correspondingly adapted, for example by extending the handle, in order to reach difficult to reach areas, for example in washing machines or coffee makers. In doing so, the test strip can be inserted into a tube, for example. Further, a sampling stick or the like may be scheduled, and such a stick can be used to collect, for example, a damp sample in the instrument and subsequently place it on the test strip.

  The invention further relates to the use of a biosensor in the form of a lateral flow test strip for the immunological detection of microorganisms in household appliances, in particular in washing machines or coffee makers. With regard to other features of this lateral flow test strip, and more particularly with respect to its use for detecting microorganisms in household appliances, it has also been indicated above.

  Finally, the present invention includes a method for detecting microorganisms in home appliances, particularly in washing machines and coffee makers. The method first involves providing a biosensor in the form of a lateral flow test strip in the above embodiment. A liquid or debris sample is taken from the household appliance and the sample is brought into contact with the lateral flow test strip. Alternatively, the lateral flow test strip can be directly immersed in, for example, a liquid in a water supply container of a home appliance. A lateral flow test is then performed to detect microorganisms optionally present in the sample based on capillary forces according to the immunological test principles described above. This detection utilizes the mobility characteristics of the microorganism-specific antibody of this test strip. This mobility characteristic varies depending on the complex formation with the microorganism to be detected. Finally, the lateral flow test strip is read. In that case, in a particularly preferred embodiment, the optical reading is performed by the user, but it is preferred that a colored detection system is provided for this test strip.

  The biosensor according to the present invention provides a unified and simple technical platform in the form of a lateral flow test strip. This technology platform can be designed as a detection method for various microorganisms by using various immunological detection reagents, in particular by using microorganism specific antibodies. This lateral flow test strip is preferably devised as a disposable article, and finishing as a lateral flow test strip makes it possible to achieve a very low cost and everyday useful embodiment.

  In addition to the use cases described above for disposable sensors in a manual test method, an automatable test can be realized by extending the equipment with sampling points or insertable cartridges. For example, a coffee maker mill can be adapted so that sampling can be automated, for example, at regular intervals and placed on a test strip for microbial analysis. Interpretation and evaluation of the test strip is also automatically performed as appropriate, and the result can be displayed, for example, on a display device, so that necessary and appropriate cleaning measures can be taken.

  Further features and advantages of the present invention are illustrated by the description of the following examples with reference to the drawings. Individual features can be realized independently or in combination with each other.

FIG. 1 shows a schematic diagram of a preferred embodiment of a lateral flow test strip as a biosensor according to the present invention. FIG. 2 shows schematic diagrams (A, B, C) of the test strip for explaining the interpretation of the test strip.

DESCRIPTION OF EXAMPLES FIG. 1 schematically shows a biosensor in the form of a lateral flow test strip 10. The test strip 10 is scheduled for immunological detection of the microorganism 50, and the microorganism-specific antibody 40 is present depending on the microorganism to be detected. A detection reagent 41 is preferably directly bound to each antibody 40, and the detection reagent 41 is, for example, a metal particle or a dye molecule. Particularly advantageously, the lateral flow test strip 10 can be used for detecting microorganisms such as filamentous fungi, filamentous fungi spores and bacteria, for example in household appliances such as washing machines and coffee makers. Furthermore, the biosensor according to the present invention as a whole can be used, for example, to examine the microbial contamination load in any device or apparatus having a place that has been wet for a long time or other places where damage to microorganisms has occurred. Furthermore, the biosensor according to the invention can in principle also be used for microbial tests on liquid or solid samples as a whole.

  The test strip 10 includes a sampling pad 11, a capillary 12, and a detection region 13 and a confirmation region 14. The microorganism-specific antibody 40 and the detection reagent 41 bound thereto can be prepared in the range of the sampling pad 11 together, for example, in a dried form. In other embodiments, a buffer can be applied, eg, in the range of the sampling pad 11, before or after placing the sample on the test strip 10, and the antibody 40 can be transferred to the test strip along with this buffer.

  When testing for damage from microorganisms, the user first begins with, for example, a washing machine (eg, from a laundry tub or laundry detergent inlet), or from a coffee maker, eg, from a water supply tank, from a ground waste, or from a mill or extractor ( Sampling is performed from the range of (Bruhgruppe). In this case, the sample may be a liquid sample or a dull sample. For example, a cotton swab can be used to facilitate the collection of a scum-like sample. Next, this sample is placed on the sampling pad 11. Alternatively, if the sample is liquid, it may be planned to immerse the test strip 10 directly in the liquid sample within the range of the sampling pad 11. In order to facilitate this immersion, particularly in areas that are difficult to reach, the test strip 10, for example, can be provided with an extension in the form of an attached stick or the like. In particular, when the sample is in the form of a dull, it may be necessary to place a running buffer in the range of the sampling pad 11. For this purpose, a buffer cartridge 30 may be provided, and the buffer can be released from the cartridge 30 manually or in an appropriately automated form as required. When the sample is in liquid form, it is generally not necessary to place a buffer. In the range of the sampling pad 11, an optionally present microorganism 50 (for example, filamentous fungus spores) is in contact with the antibody 40. When these microorganisms 50 and the antibody 40 are specifically bound (antigen / antibody binding), a complex is formed as a result. These complexes exhibit different flow characteristics than the free antibody 40, thereby causing separation within the capillary 12. Free antibody 40 flows to a greater extent or more rapidly to some extent than the complex of antibody 40 and microorganism 50. At that time, the free antibody 40 reaches the confirmation region 14 which is further away. The complex of the antibody 40 and the filamentous fungus spore 50 reaches the detection region 13 only. This flow behavior can be visually recognized by the color reaction generated by the detection reagent 41 in each region 13 or 14. When metal particles are used as the detection reagent 41, for example, discoloration may occur due to the metal particles in the detection region 13 and / or the confirmation region 14. When a dye is used as the detection reagent 41, for example, a direct or indirect color reaction can occur in the detection region 13 and / or the confirmation region 14. Can be deciphered by physical inspection. Depending on in which region the color reaction is detectable, the user can read the presence or absence of microorganisms in the sample. In doing so, the user obtains qualitative detection or semi-quantitative or quantitative results, in which case the test strip 10 equipment and in particular the reagent concentration, in particular the antibody 40 concentration, the microorganism to be detected. It is preferable that the threshold value of the problematic concentration is considered. In some cases, an optical device such as a camera can be used for interpretation.

  In one particularly preferred embodiment, the antibody is bound directly or directly to the dye or metal particle, so that the presence of the antibody in each reading region (detection region and / or confirmation region) is colored. It can be detected directly as a reaction. In other embodiments, it is also conceivable that a color reaction occurs for the first time in combination with a further detection reagent, for example by a known enzymatic detection reaction. In this case, the test strip must incorporate the corresponding detection reagent, in particular in the region of the detection region and possibly also in the region of the confirmation region. Furthermore, for example, fluorescent dyes can be used.

  The confirmation area 14 is finished in particular so that the reaction can be detected in this confirmation area only if the test strip 10 is used correctly, in which case there is an excess on the test strip in this confirmation area. The color reaction caused by the free antibody and the detection reagent bound to the free antibody becomes visible in the above-described manner, for example.

  FIG. 2 shows the possible results when a microbial contamination load test is performed on the basis of FIGS. A, B and C of a test strip 200 according to the invention. Depending on the location and number of linear color reactions in the ranges 210, 220, 230, the results can be read. In the case of A, a single colored line is observed in range 210. This line appears in the confirmation area of the test strip 200. This confirmation region is located farthest from the sampling pad (not shown in detail in FIG. 2). In this case, the corresponding microorganism could not be detected (result is negative). In the case of B, in the range 220, colored lines are recognized not only in the confirmation region but also in the detection region closer to the sampling pad (not shown). In this case, the test result was positive and thus the microorganism could be detected. In the case of C, in the range 230, a colored line can be recognized only within the detection region. This result is invalid because no line appears in the confirmation area.

  By using such a biosensor in the form of a lateral flow test strip according to the present invention, for example, the presence or absence of a microbial contamination load in a washing machine or coffee maker can be easily examined by the user himself.

  Prior to the cleaning action to be taken on a regular basis, the user can, for example, determine whether a microbial contamination load actually exists and needs cleaning. In this way, for example, the implementation of cleaning measures that often involve the use of aggressive cleaning substances can be reduced to the extent necessary.

Claims (13)

  A biosensor for immunologically detecting a microorganism (50), the biosensor being finished in the form of a lateral flow test strip (10) and having a microorganism-specific antibody (40), When the antibody (40) and the microorganism (50) to be detected form a complex, the mobility characteristic of the antibody (40) changes.   The biosensor according to claim 1, characterized in that at least one detection reagent (41) is bound to the microorganism-specific antibody (40).   The biosensor according to claim 1 or 2, characterized in that the detection reagent (41) contains metal particles or dye molecules.   The biosensor according to any one of claims 1 to 3, wherein the biosensor is intended to be used for detection of filamentous fungi and / or spores and / or bacteria of the filamentous fungi.   The biosensor according to any one of claims 1 to 4, characterized in that the microorganism-specific antibody (40) corresponds to a surface molecule of the microorganism (50) to be detected.   The biosensor according to any one of claims 1 to 5, wherein the antibody (40) corresponds to β-glucan of spores of filamentous fungi.   The biosensor according to any one of claims 1 to 6, characterized in that the lateral flow test strip (10) is a disposable article.   8. The lateral flow test strip (10) according to any one of the preceding claims, characterized in that it comprises a sampling pad (11) and / or a capillary (12) and / or at least one detection region (13). The biosensor according to item 1.   Biosensor according to any one of the preceding claims, characterized in that the lateral flow test strip (10) comprises a confirmation region (14).   10. The biosensor according to claim 1, wherein at least one buffer cartridge (30) is incorporated in the lateral flow test strip (10).   Biosensor according to any one of the preceding claims, characterized in that it is adapted for immunological detection of microorganisms (50) in household appliances, in particular in washing machines or coffee makers.   12. Biosensor in the form of a lateral flow test strip (10) according to any one of claims 1 to 11, for immunological detection of microorganisms (50) in household appliances, in particular in washing machines or coffee makers. Use of. A method for detecting microorganisms in household appliances, in particular in washing machines or coffee makers,
Providing a biosensor in the form of a lateral flow test strip (10) according to any one of the preceding claims;
Contacting a liquid sample or a damp sample taken from the household appliance with the lateral flow test strip (10);
Reading the lateral flow test strip (10).

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