State of the art

Currently, soil enzymatic activities are measured in the laboratory using destructive biochemical laboratory incubations that disperse soil in an aqueous solution. For measuring soil enzymatic activity basically two methods are known, which in scientific literature are referred to as slurry assay and zymography respectively. The drawbacks of current systems and methods for measuring soil enzymatic activity are described in detail, for example in scientific literature 1 (GERMAN, D. P. et al., Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies. Soil Biology and Biochemistry, Issue 43, 2011 , pages 1387-1397) and scientific literature 2 (DENG, S. et al., Comparison and standardization of soil enzyme assay for meaningful data interpretation, Journal of Microbiological Methods, Issue 133, 2017, pages 32 - 34).

Moreover, the known methods, i.e., the classical slurry assay and the zymography have limitations with regard to the soil sampling, where protocols widely vary between experiments, which then results in an unreliable comparison between studies. Furthermore, the collection, preparation, and storage of the soil samples are causing a significant alteration to enzyme activity as described in scientific literature 3 (DE FOREST, J.L., The influence of time, storage temperature, and substrate age on potential soil enzyme activity in acidic forest soils using MUB- linked substrates and l-DOPA, Soil Biology and Biochemistry, Issue 41 , 2009, pages 1180 - 1186) and scientific literature 4 (DADENKO, E. V. et al., Changes in the enzymatic activity of soil samples upon their storage, Eurasian Soil Science, Issue 42, 2009, pages 1380 -1385). Finally, the enzyme activity measurements of the soil samples are labor-intensive since the known methods require several optimization and control experiments prior to the measurements as described in detail in scientific literature 1 , scientific literature 5 (DICK, W.A., Development of a Soil Enzyme Reaction Assay, Methods of Soil Enzymology, Volume 9, 2011 , Chapter 4), scientific literature 6 (RAZAVI, B. S. et aL, 2019. Soil zymography: Simple and reliable? Review of current knowledge and optimization of the method, Rhizosphere, Issue 11 , 2019:100161 .2019). The limitations of known methods for measuring soil enzymatic activity can be summarized as follows: The known methods are destructive and often alter the pH-value of the soil with buffers, and thus, do not represent the in-situ enzyme activity. This applies especially for the slurry assay, since it requires the mixing of the soil with water. Furthermore, the enzymatic activity rate can only be measured after the addition of water and/or buffer liquid in big excess as described in scientific literature 6 and scientific literature 7 (MARX, M.-C. et aL, A microplate fluorimetric assay for the study of enzyme diversity in soils, Soil Biology and Biochemistry, Issue 33, pages 1633 - 1640). Moreover, according to scientific literature 7 the known methods may deliver imprecise, often too low results, which are caused by interferences of the chromogenic and fluorogenic product with soil organic matter when using the classical slurry assays, and because of insufficient contact between the membrane and the soil when using zymography. In addition, the classical slurry assay using fluorometric detection requires expensive lab equipment. Finally, the classical slurry assays and the zymography are time-intensive, from preparing the chemicals to the preparation of the plate or of the membrane, the measurements, and the analysis. For instance, it takes at least one whole day to measure the enzymatic activity using the classical slurry assay and analyze the data. In summary known methods for measuring soil enzymatic activity have limited use due to many methodological constraints, like artificial measurement conditions, the influence of transportation and storage on the samples, intensive labor, tedious analysis and high costs. These difficulties prevent scientists, governments, farmers, and gardeners from monitoring soil enzyme activities, which could aid decision-making, with a reasonable effort. Disclosure of the invention

An objective of the invention is, in particular, to provide a measuring system and a method for measuring soil enzymatic activity with improved characteristics regarding an efficiency. The objective is achieved, according to the invention, by the features of claim 1 and claim 14, while advantageous implementations and further developments of the invention may be gathered from the dependent claims.

The invention is based on a measuring system for measuring soil enzymatic activity, with at least one measuring device comprising:

- a reaction unit with at least one reaction element containing at least one substrate embedded in a gel, the reaction element being configured for directly contacting a soil sample and enabling at least one chemical reaction process between the substrate and at least one type of enzyme in the soil sample;

- a detection unit for detecting at least one optically active reaction product released by the chemical reaction process; and

- a processing unit for processing of data detected by means of the detection unit.

It is proposed that the measuring device is configured for measuring at least one enzymatic activity parameter, in particular an enzymatic activity rate, of the enzyme in the soil sample directly during the chemical reaction process.

By means of the present invention advantageously a highly efficient measuring system for measuring soil enzymatic activity can be provided. In particular measurement results can be obtained faster and cheaper compared to state of the art. Moreover, an accuracy can be improved. In particular measurement results can be obtained in a more reliable manner and can be more easily compared to each other. A further advantage of the measuring system according to the present invention is that soil enzymatic activity is measured under natural conditions.

Neither storage nor incubation of the soil sample is required and the measurement can be done directly in the field. Compared with zymography the rate of fluorescence increase can be quantified, while zymography allows only one time point measurements. The measuring system includes the at least one measuring device. In the simplest case the measuring device forms the measuring system. However, the measuring system can also include a plurality of measuring devices. Moreover, the measuring system may include components and/or units and/or elements which are not part of the measuring device(s). For instance, the measuring system preferably comprises at least one server including a database for storing data related to the measuring system. Preferably the measuring system also includes an app to operate on an external device in communication with the database. In the context of the present document an “external device” is to mean a device, which is not part of the measuring system as such, and which is capable of wireless communication, such as a smartphone and/or a tablet computer and/or a smartwatch and/or a laptop and/or computer and/or the like.

The measuring device comprises the reaction unit with the at least one reaction element. The reaction unit may comprise a plurality of reaction elements, which can be identical in their basic geometrical structure, but may differ for example with regard to the at least one substrate contained therein. The at least one substrate contained in the reaction element can be a luminescent and/or chromogenic and/or colorogenic compound, for example p-nitrophenol. Advantageously, however, the at least one substrate contained in the at least one reaction element is a fluorogenic compound. Possible suitable fluorogenic compounds which could be used as a substrate are, without being limited to those, for example 4-Methylumbelliferyl N-acetyl-[3-D-glucosaminide (CAS 37067-30-4) and/or 4-Methylumbelliferyl [3-D-glucopyranoside (CAS18997-57-4) and/or 4- Methylumbelliferyl phosphate (CAS 3368-04-5) and/or 4-Methylumbelliferyl-[3-D- xylopyranoside (CAS 6734-33-4) and/or L-Leucine-7-amido-4-methylcoumarin hydrochloride. The reaction element may contain different kinds of substrates for enabling different kinds of chemical reaction processes with different types of enzymes in the soil sample. The substrate or substrates contained in the reaction element is/are embedded in a gel. For example, but not limited to, the gel can be made from agarose and water. Advantageously the gel is at least substantially transparent. The term "at least substantially transparent” in this context is to be understood as the property of allowing light to pass through the material without appreciable scattering of light. The gel can for example be prepared by mixing agarose powder, which has a low gelling temperature, with Milli-Q-water, wherein the mixture is boiled while mixing. Afterwards the mixture can be cooled down to 65 C and the desired substrate can be added into the solution. The substrate embedded in the gel then can then be transferred to the reaction element while still hot so that it can solidify in the reaction element, which is closed off on one side with a foil or a lid or a liquid barrier or the like. After the gel is solidified the other side of the reaction element is also closed off with a foil or a lid or a liquid barrier or the like. The whole process is preferably conducted under sterile conditions to avoid contamination of the reaction element with enzymes before contact with the soil sample to be measured.

When the soil sample is directly contacted by the reaction element the at least one chemical reaction process between the substrate and at least one type of enzyme in the soil sample is enabled. It is however to be noted that in the event there are no enzymes present in a particular soil sample, which are targeted by a particular substrate contained in the reaction element, of course the intended chemical reaction process cannot be enabled. All methods that measure enzyme activity, including the one carried out by the measuring system and/or used for the method for measuring soil enzymatic activity according to the present invention, are based on the same principle: a functional group is bound to a chromogenic or fluorogenic compound which after the enzymatic reaction is released and quantified by light absorption, in case of a chromogenic compound, or by fluorescence measurement, in case of a fluorogenic compound. The exact chemical and/or physical processes underlying this principle are known to the person skilled in the art, or may be gathered from scientific literature 1 to 7, the contents of which are incorporated herein be reference.

The measuring device further comprises the detection unit for detecting at least one optically active reaction product released by the chemical reaction process. In the present document, an “optically active reaction product” is to be understood as at least one compound, which is released by the chemical reaction process and which, in comparison to a state before the chemical reaction process, can be detected due to its optical properties, for example its color and/or its light absorption properties and or its fluorescence and/or luminescence. The detection unit in particular comprises at least one lighting element, which is configured for emitting light at a certain wavelength, advantageously at a wavelength between 260 nm and 360 nm, especially advantageous at a wavelength between 280 nm and 340 nm, preferably at a wavelength between 300 nm and 330 nm, especially preferred at a wavelength of 310 nm. The at least one lighting element of the detection preferably is embodied as an LED. However, in the alternative any other type of light source, which is capable of emitting light at the above-mentioned wavelength, can also be used as a lighting element. In an assembled state of the measuring device the lightning element(s) of the detection unit are arranged at a fixed height towards the reaction element, wherein the height is selected in way to allow the best measuring results. The lighting element(s) is/are configured to illuminate the upper side of the reaction element at regular intervals, e.g., every 3 minutes or every 2 minutes, preferably every minute, and for a fixed time period, ranging between 1 second and 10 seconds. The detection unit comprises at least one detection element, which is configured to detect the light intensity of the reaction element during the time period of illumination by the lighting element. The detection element is preferably embodied as a camera. However, also other types of photodetectors, such as photodiodes and/or phototubes and/or photomultiplier tubes and/or photoresistors and/or active-pixel sensors and/or charge-coupled devices and/or the like, may be used as detection elements in the alternative or in addition.

The measuring device further comprises the processing unit for processing of data detected by means of the detection unit, which for this purpose is electrically connected to the at least one detection element of the detection unit. The processing unit comprises at least one computational unit, such as a microprocessor and or ASIC or the like, and at least one memory unit. The detection unit is configured to determine the at least one enzymatic activity parameter, in particular the enzymatic activity rate, of the at least one type of enzyme, by extracting measuring points out of data detected by means of the detection unit, e.g., extracting RGB values of pictures taken by the detection unit, and comparing them with reference points, which may be stored in the memory unit or which preferably are gathered by the detection unit from at least one optically active reaction product, which is not bound to any substrate, and which can also be contained in the reaction element in known concentration for calibration purposes.

The measuring device is configured for measuring at least one enzymatic activity parameter of the at least one type of enzyme in the soil sample during the chemical reaction process. The at least one enzymatic activity parameter preferably is an enzymatic activity rate. However, in the broadest sense of the invention the at least one enzymatic activity parameter may refer to only one measuring point extracted by the processing unit, which allows to determine whether a certain enzyme targeted by the at least one substrate contained in the reaction element is present at all in the soil sample or not. The at least one activity parameter of the at least one type of enzyme in the soil sample is measured during the chemical reaction process and is issued by the processing unit advantageously after one hour, especially advantageously after 50 minutes, preferably after 45 minutes and especially preferred after 40 minutes, from the beginning of the at least one chemical reaction process.

In the context of the present document, “configured” and/or “intended” is to mean specifically programmed, designed and/or equipped. By an object being configured and/or intended for a certain function, it is to be understood that the object fulfills and/or implements this certain function in at least one application state and/or operating state. Moreover, it should be noted, that in the present document, number words, such as “first”, “second” and “further”, which precede certain terms, serve merely to distinguish objects and/or an assignment between objects and do not imply an existing total number and/or ranking of objects and/or process steps. In particular, a “second” or “further” object and/or process step does not necessarily imply the existence of a “first” object and/or process step.

Furthermore, it is proposed that the reaction unit is configured for keeping the composition of the soil sample at least substantially unaltered during the chemical reaction process. Thereby advantageously an accuracy of soil enzymatic activity measurements can be improved. In this context, the statement that the reaction unit is configured for keeping the composition of the soil sample "at least substantially unaltered" should be understood to mean that composition of the soil sample before and after a measurement of soil enzymatic activity carried out with the measuring system according to the present invention is essentially the same, apart from the enzyme level in the soil sample, which may changes due to migration of enzymes into the reaction element of the reaction unit, apart from a level of substrates, which possibly migrate out of the reaction element into the soil sample during the chemical reaction process and apart from a moisture level of the soil sample, as the gel contained in the reaction element can moisten the soil sample at the contact areas with the soil sample. Preferably the reaction unit is also configured for keeping the pH-value of the soil sample at least substantially unaltered during the chemical reaction process. In this context, the statement that the reaction unit is configured for keeping pH-value of the soil sample "at least substantially unaltered" should be understood to mean that the pH-values of the soil sample measured before and after a measurement of soil enzymatic activity carried out with the measuring system according to the present invention is essentially the same within the scope of usual measurement inaccuracies.

Moreover, it is proposed that the at least one reaction element is embodied as a membrane. Thereby advantageously a good substance exchange between the soil sample and the reaction element can be achieved, as enzymes contained in the soil sample are able to migrate into the reaction element, if the reaction element is embodied as a membrane. In an advantageous embodiment of the invention, it is proposed that the reaction element comprises at least one reaction well containing the substrate. In that way, advantageously the reaction element can be easily equipped with the substrate. In particular the substrate embedded in the gel can be easily added in precisely specified quantities into the reaction element, e.g., by transferring the substrate embedded in the gel into the reaction well(s) before solidification by means of a pipetting robot. The reaction element comprises a base body in which the at least one reaction well is arranged. Preferably the reaction well is continuous from a top to a bottom of the base body of the reaction element. The reaction element may contain a plurality of reaction wells, which can be arranged spaced apart from one- another in the base body of the reaction element. In case the reaction element comprises a plurality of reaction wells, at least some, in particular each, of the reaction wells can contain a different substrate targeting a different type of enzyme in the soil sample. Thereby advantageously an efficiency can further be improved, since different types of enzymes contained in the soil sample can be measured at the same time. In the alternative or additional it is however also conceivable that at least some of the reaction wells of the reaction element contain the same substrate, so that a reliability of the measurement can be increased and a non- uniform distribution of enzymes within the soil sample can be taken into account.

In addition, it is proposed that the reaction element comprises at least three calibration wells which contain different concentrations of at least one optically active reaction product not bound to any substrate and which are configured for calibration of the detection unit. Thereby advantageously a reliability of the measurement can be further improved. In particular differences in the spectral background of different soil samples can be taken into account, when the reaction element comprises at least three calibration wells which contain different concentrations of at least one optically active reaction product not bound to any substrate and which are configured for calibration of the detection unit. The at least one optically active reaction product contained in the calibration wells is a compound which is released by the chemical reaction process between at least one type of enzyme in the soil sample and the substrate targeting this type of enzyme. The at least one optically active reaction product contained in the calibration wells and released by the chemical reaction process can, without being limited to those compounds, for example be 4-Methylumbelliferon (MUF) and/or 7- Amino-4-Methylcoumarin (AMC). Preferably the processing unit is configured to create at least one calibration curve based on the light intensities of the calibration wells detected by the detection unit.

Furthermore, it is proposed that the reaction unit comprises at least two interchangeable reaction elements, each of which comprises a unique identifier, which can be scanned by an external device. By means of this embodiment advantageously an operating convenience can be enhanced. Preferably the unique identifier can also be scanned by the detection unit. For example, during preparation of the reaction elements the type(s) of substrate(s) contained in the reaction well(s) and the type(s) and concentration(s) of the optically active reaction product(s) contained in the calibration wells can be assigned to each reaction element by scanning the unique identifier and storing the respective data in a database. The detection unit scans the unique identifier during the measurement and the processing unit can then directly access this data und use it to determine the at least one enzymatic activity parameter. The unique identifier may be embodied as an optically unambiguously identifiable feature, such as a QR-Code or Barcode or the like. However, it is also conceivable that the unique identifier may be configured for identification by means of electronic signals and may be embodied as an RFID-tag or the like. Moreover, the unique identifier could also be embodied as any other type of identifier suitable for unambiguously identifying the respective reaction element.

Moreover, it is proposed that the measuring system comprises a sampling unit with at least one soil container to keep the soil sample, the soil container comprising at least one fixing element by means of which at least one sieve of the sampling unit can be detachably attached. Thereby ease of preparation of the soil sample can be achieved. The sieve can be used for directly sampling soil into the soil container, thereby the soil sample being homogenized and foreign objects, such as roots, stones and the like, may be removed by means of the sieve. Preferably the sieve comprises a plurality of holes, each approximately 4 mm in diameter. However, smaller or larger diameters of the holes of the sieve are of course also conceivable. The sieve preferably comprises at least one cooperating fixing element for detachable attachment to the soil container. The fixing element of the soil container and the cooperating fixing element of the sieve could be configured to form a force-fit and/or form-fit connection, e.g., a screw connection or the like. Especially preferred the fixing element of the soil container and the cooperating fixing element of the sieve are embodied as cooperating latching elements, so that the connection can be made and released advantageously without additional tools. Preferably the sampling unit also comprises a lid for each of the soil containers, which also comprises a cooperating fixing element, which is substantially identical to the cooperating fixing element of the sieve. This allows the soil container to be securely closed after the soil sample has been taken, in particular in cases when the measurement of the soil enzymatic activity is not carried out directly on-site.

In addition, it is proposed that the reaction element comprises at least one cooperating fixing element for a detachable connection with the fixing element of the soil container. By means of this embodiment advantageously an operating convenience can be further increased. Furthermore, advantageously it can be assured that the soil sample is sufficiently and evenly contacted by the reaction element when the reaction element is connected to the soil container. Preferably the cooperating fixing element of the reaction element is substantially identical to the cooperating fixing element of the sieve and hence preferably is also embodied as a latching element, such that the reaction element can be easily clicked on the soil container.

Furthermore, it is proposed that sampling unit comprises a plurality of soil containers, each of which comprises a unique identifier, which can be scanned by an external device. Thereby advantageously an operational convenience can be further improved, in particular labeling errors can be avoided. Just like the unique identifiers of the reaction elements the unique identifiers of the soil container can be embodied as optically unambiguously identifiable feature, such as a QR-Code or Barcode or the like, or as an RFID-tag or the like or as any other type of identifier suitable for unambiguously identifying the respective reaction element. During sampling of the soil sample into a soil container of the sampling unit, the user may retrieve the exact position information of the location where the sample was taken, e.g., GPS-coordinates or the like, e.g., by aid of an external device, such as his smartphone, which then can be assigned to the unique identifier of the soil container.

In further advantageous embodiment of the invention, it is proposed that the sampling unit comprises a holding unit for holding a plurality of soil containers, the holding unit including a rotating drum configured for subsequently bringing different soil samples in each of the soil containers into contact with at least one reaction element of the reaction unit. By means of such an embodiment advantageously an efficiency, particularly a time efficiency, can be further increased, in particular if a large number of samples are to be analyzed.

Moreover, it is proposed that the measuring system comprises a communication unit for wireless communication between the processing unit and at least one external device. Thereby an operational convenience can pe improved. By means of the communication unit data can be exchanged between the measuring system and the external device wirelessly and bidirectional, e.g., by means of Bluetooth, NFC, WIFI or any other suitable wireless communication standard. For example, data, such as position information, retrieved on-site, can be easily sent to the measuring system. On the other hand, also results of the measurements carried out with the measuring device can be easily retrieved by the external device, even remotely, when the measuring system comprises a communication unit for wireless communication between the processing unit and at least one external device. The measuring system may comprise exactly one measuring device. In another advantageous embodiment it is however proposed that the measuring system comprises at least one further measuring device, which is at least substantially identical to the measuring device and which is stackable with the measuring device. Thereby advantageously an efficiency can be further increased. In this embodiment the measuring device and the further measuring device may in particular be miniaturized, such that they are portable and can be used directly onsite.

The invention further relates to a reaction element for a measuring system according to any of the previously described embodiments. The reaction element is in particular distinguished by its above-mentioned advantageous features with regard to reliable and accurate measuring of soil enzymatic activity.

The invention is further based on a method for measuring soil enzymatic activity, in particular with a measuring system according to any one of the previously described embodiments, wherein at least one reaction element containing at least one substrate embedded in a gel is brought into direct contact with a soil sample to initiate at least one chemical reaction process between at least one type of enzyme in the soil sample and the substrate and wherein at least one optically active reaction product released by the chemical reaction process is detected.

It is proposed that at least one enzymatic activity parameter, in particular an enzymatic rate, of the enzyme in the soil sample is measured directly during the chemical reaction process. Thereby advantageously a very efficient and reliable method for measuring soil enzymatic activity can be provided.

In addition, it is proposed that a plurality of measuring devices are successively brought into direct contact with soil samples at different locations spaced apart from each other and wherein in each soil sample at least one enzymatic activity parameter is measured simultaneously. Thereby advantageously an efficiency, in particular a time-efficiency, can be further increased. The measuring system according to the invention and the method for measuring soil enzymatic activity according to the invention shall herein not be limited to the applications and implementations described above. In particular, in order to fulfill a certain functionality that is described herein, the measuring system according to the and/or the method for measuring soil enzymatic activity according to the invention may comprise a number of individual elements, components and units as well as method steps that differ from a number given here. Moreover, concerning value ranges given in the present disclosure, values within the limits mentioned shall also be considered to be disclosed and to be usable as applicable.

Drawings

Further advantages may become apparent from the following description of the drawings. In the drawings three exemplary embodiments of the invention are shown. The drawings, the description and the claims contain a plurality of features in combination. Someone having ordinary skill in the art will purposefully also consider the features separately and will find further expedient combinations.

It is shown in:

Fig. 1 a measuring system with a measuring device in a schematic perspective view,

Fig. 2 a schematic sectional view of the measuring device,

Fig. 3 a reaction element of a reaction unit of the measuring system in a schematic top view,

Fig. 4 a sampling unit of the measuring system with a soil container and a sieve in a schematic perspective view,

Fig. 5 the soil container and the reaction element in a schematic perspective view,

Fig. 6 three schematic diagrams to illustrate a mode of operation of the measuring device, Fig. 7 a schematic flow chart of a method for measuring soil enzymatic activity,

Fig. 8 a schematic illustration of a measuring system in another embodiment of the invention and

Fig. 9 a schematic illustration of a measuring system in another embodiment of the invention.

Description of the exemplary embodiments

If there is more than one specimen of a certain object, only one of these is given a reference sign in the figures and in the description. The description of this specimen may be correspondingly transferred to the other specimens of the object. Furthermore, the figures are schematic and not true-to-scale.

Figure 1 shows a measuring system 10a for measuring soil enzymatic activity in a schematic perspective view. The measuring system 10a includes at least one measuring device 12a. The measuring device 12a comprises a reaction unit 14a with at least one reaction element 16a (cf. figure 3) containing at least one substrate (not shown) embedded in a gel 18a (cf. figure 3), the reaction element 16a being configured for directly contacting a soil sample (not shown) and enabling at least one chemical reaction process between the substrate and at least one type of enzyme in the soil sample. The measuring device 12a further comprises a detection unit 20a for detecting at least one optically active reaction product (not shown) released by the chemical reaction process. Furthermore, the measuring device 12a comprises a processing unit 22a for processing of data detected by means of the detection unit 20a.

The measuring device 12a includes a housing 104a in which, among others, the reaction unit 14a, the detection unit 20a and the processing unit 22a are housed. The reaction unit 14a, the detection unit 20a and the processing unit 22a will be described in more detail later. Furthermore, the measuring device 12a comprises a display unit 106a, which is connected to the processing unit 22a and which is configured to display results of measurements carried out with the measuring device 12a.

Moreover, the measuring system 10a comprises a communication unit 72a for wireless communication between the processing unit 22a and at least one external device 48a. According to the present embodiment of the invention the external device 48a is embodied as a smartphone. However, also other types of external devices (not shown), e.g., tablet computers or the like, are conceivable for use in combination with the measuring system 10a. By means of the communication unit 72a data can be exchanged between the measuring device 12a and the external device 48a wirelessly and bidirectionally, e.g., by means of Bluetooth, NFC, WIFI or any other suitable wireless communication standard.

In figure 2 the measuring device 12a is shown in a schematic sectional view.

The measuring system 10a comprises a sampling unit 50a. The sampling unit 50a comprises at least one soil container 52a to keep the soil sample. The soil container 52a can be inserted into the housing 104a by means of a drawer 108a. When the soil container 52a is inserted into the housing 104a it is arranged below the detection unit 20a. The reaction element 16a of the reaction unit 14a is then arranged above the soil container 52a such that it is in direct contact with the soil sample contained therein.

The detection unit 20a comprises a holder 110a. The detection unit 20a comprises at least one lighting element 114a, which can be embodied as an LED for example, and which is configured to emit light at a certain wavelength, advantageously between 300 nm and 320 nm. The detection unit 20a comprises at least one light sensor element 116a, which can be embodied as a camera or a photodiode or any other suitable element for detecting light and which is connected with the processing unit 22a. The processing unit 22a comprises at least one microprocessor or any other suitable type of processor for processing data detected by the detection unit 20a. The at least one lighting element 114a and the least one sensor element 116a are fixed to the holder, such that a suitable distance towards the reaction element 16a allowing the best measurement results can be kept.

Figure 3 shows the reaction element 16a of the reaction unit 14a in a schematic top view. The reaction element 16a containing at least one substrate (not shown) embedded in a gel 18a. The reaction element 16a being configured for directly contacting a soil sample (not shown) and enabling at least one chemical reaction process between the substrate and at least one type of enzyme in the soil sample. In order to enable the at least one chemical reaction process a bottom of the reaction element 16a is pressed against the soil sample. The reaction unit 14a is configured for keeping the composition of the soil sample at least substantially unaltered during the chemical reaction process.

In the present embodiment the at least one reaction element 16a is embodied as a membrane. The reaction element 16a comprises at least one reaction well 24a containing the substrate embedded in the gel 18a. The gel 18a is at least substantially transparent to light and for example can be made of agarose and water.

In the present embodiment the reaction element 16a comprises a plurality of reactions wells 24a, 26a, 28a, 30a, 32a. Three reaction wells 24a1 , 24a2, 24a3 of a first group each contain a first type of substrate embedded in the gel 18a. Three reaction wells 26a1 , 26a2, 26a3 of a second group each contain a second type of substrate embedded in the gel 18a. Three reaction wells 28a1 , 28a2, 28a3 of a third group each contain a third type of substrate embedded in the gel 18a. Three reaction wells 30a1 , 30a2, 30a3 of a fourth group each contain a fourth type of substrate embedded in the gel 18a. Three reaction wells 32a1 , 32a2, 32a3 of a fifth group each contain a fifth type of substrate embedded in the gel 18a. Hence, the reaction element 16a in the present embodiment comprises 15 reactions wells 24a, 26a, 28a, 30a, 32a in total comprising five different types of substrates targeting five different types of enzymes, which may be contained in the soil sample. For a person skilled in the art, it will however become apparent that, depending on the number of different types of enzymes to be targeted, also a different, i.e., a lower or higher, number of reaction wells 24a, 26a, 28a, 30a, 32a will be sufficient to realize the general idea of the present invention.

Moreover, the reaction element 16a comprises at least three calibration wells 34a, 36a, 38a, 40a, 42a which contain different concentrations of at least one optically active reaction product not bound to any substrate and which are configured for calibration of the detection unit. In the present embodiment of the invention the reaction element 16a comprises two calibration wells 34a1 , 34a2 of a first group, which contain a first optically active reaction product in different concentrations embedded in the gel 18a. The reaction element 16a comprises two calibration wells 36a1 , 36a2 of a second group, which contain a second optically active reaction product in different concentrations embedded in the gel 18a. The reaction element 16a further comprises two calibration wells 38a1 , 38a2 of a third group, which contain a third optically active reaction product in different concentrations embedded in the gel 18a. Moreover, the reaction element 16a comprises two calibration wells 40a1 , 40a2 of a fourth group, which contain a fourth optically active reaction product in different concentrations embedded in the gel 18a.

Furthermore, the reaction element 16a comprises two calibration wells 42a1 , 42a2 of a fifth group, which contain a fifth optically active reaction product in different concentrations embedded in the gel 18a.

In the present embodiment each of the reaction wells 24a, 26a, 28a, 30a, 32a and each of the calibration wells 34a, 36a, 38a, 40a, 42a are filled using the same gel 18a but containing different kinds of substrates or optically active reaction products as subsequently described. The reaction wells 24a, 26a, 28a, 30a, 32a and the calibration wells 34a, 36a, 38a, 40a, 42a are continuous from a top to a bottom of the reaction element 16a and the height of the gel does not exceed 4 mm in each case, such that quenching of the light, which is provided by the at least one lighting element 114a (cf. figure 2) can be omitted. In table 1 below examples of possible substrates, which can be embedded in the gel 18a are listed with their abbreviations and their full chemical names:

Table 1 : Examples of possible substrates and optically active reaction products

For example, the three reaction wells 24a1 , 24a2, 24a3 of the first group may each contain NAG as a substrate embedded in the gel 18a. The three reaction wells 26a1 , 26a2, 26a3 of the second group may each contain GLS as a substrate embedded in the gel 18a. The three reaction wells 28a1 , 28a2, 28a3 of the third group each may each contain MUP as a substrate embedded in the gel 18a. The three reaction wells 30a1 , 30a2, 30a3 of the fourth group may each contain MUX as a substrate embedded in the gel 18a. The three reaction wells 32a1 , 32a2, 32a3 of the fifth group may each contain LAP/LEU as a substrate embedded in the gel 18a. Furthermore, in table 1 above the target groups of enzymes, which are targeted by the respective substrates and the equivalent natural substrates of the mentioned substrates are listed. Moreover, table 1 lists abbreviations of the optically active reaction products, which can be obtained from the respective substrates in the chemical reaction processes with the targeted types of enzymes in the soil sample. In the chemical reaction process with the target group of enzymes the first four types of substates mentioned in table 1 release 4- Methylumbelliferon (MUF) as an optically active reaction product. A chemical reaction process between the substrate LAP/LEU with the target group of enzymes releases 7-Amino-4-Methylcoumarin (AMC). Accordingly, the two calibration wells 34a1 , 34a2 of the first group, the two calibration wells 36a1 , 36a2 of the second group, the two calibration wells 38a1 , 38a2 of the third group and the two calibration wells 40a1 , 40a2 of the fourth group may each contain MUF in different concentrations. In the two calibration wells 42a1 , 42a2 of the fifth group AMC may contain AMC in different concentrations.

In figure 4 the sampling unit 50a of the measuring system 10a is shown in schematic perspective view with the soil container 52a and a sieve 58a.

The soil container 52a comprises at least one fixing element 54a by means of which the at least one sieve 58a of the sampling unit 50a can be detachably attached. In the present embodiment of the invention the soil container 52a comprises a first fixing element 54a and second fixing element 56a. The sieve 58a comprises a first cooperating fixing element 76a and a second cooperating fixing element 78a. The first cooperating fixing element 76a of the sieve 58a corresponds to the first fixing element 54a of the soil container 52a and the second cooperating fixing element 78a of the sieve 58a corresponds to the second fixing element 56a such that the sieve 58a can be detachably clicked on the soil container 52a. The sieve 58a of the sampling unit 50a is intended for preparation of the soil sample. When the sieve 58a is attached to the soil container 52a a user can sample soil directly into the soil container 52a, thereby the soil sample being homogenized and foreign objects, such as roots, stones and the like, may be removed by means of the sieve 58a.

Figure 5 shows the reaction element 16a and the soil container 52a in schematic perspective view.

The reaction element 16a comprises at least one cooperating fixing element 60a for a detachable connection with the fixing element 54a of the soil container 52a. In the present embodiment of the invention, just like the sieve 58a, the reaction element 16a comprises a first cooperating fixing element 60a corresponding to the first fixing element 54a of the soil container 52a and a second cooperating fixing element 62a corresponding to the second fixing element 56a of the soil container 52a such that the reaction element 16a can be clicked on the soil container 52a in order to directly contact the soil sample disposed therein after the sieve 58a has been removed.

The reaction unit 14a comprises a plurality of at least two interchangeable reaction elements 16a. However, for the sake of simplicity, in figure 5 only one of the interchangeable reaction elements 16a is shown. All reaction elements 16a of the reaction unit 14a are identical in their basic geometrical structure, but may differ, for example, in the number of reaction wells 24a, 26a, 28a, 30a, 32a and/or number of calibration wells 34a, 36a, 38a, 40a, 42a, and/or in the types of substrates filled in the reaction wells 24a, 26a, 28a, 30a, 32a and or in types of optical products and/or concentrations of optical products contained inside the calibration wells 34a, 36a, 38a, 40a, 42a.

Each reaction element 16a of the reaction unit 14a comprises a unique identifier 46a, which can be scanned by the external device 48a. In the present embodiment of the invention the unique identifier is embodied as a QR-Code, which can be scanned by a camera of the external device 48a. However, in the alternative also other types of unique identifiers, for example bar-codes, RFID-tags or any other type of identifier suitable for unambiguously identifying the respective reaction element 16a, are conceivable without departing from the scope of the present invention.

Furthermore, the sampling unit 50a comprises a plurality of soil containers 52a, each of which comprises a unique identifier 66a, which can be scanned by the external device 48a. Again, for the sake of simplicity, in figure 5 only one of the soil containers 52a is shown. All soil containers 52a of the sampling unit 50a are at least substantially identical, of course with the exception of their respective unique identifier 66a, which in the present embodiment of the invention also is embodied by way of example is also embodied as QR-Code, but which in the alternative also could be embodied as any other type of identifier suitable for unambiguously identifying the respective soil container 52a. Each soil container 52a of the sampling unit 50a may be used for sampling of different soil samples, wherein the unique identifier may help to distinguish these different soil samples afterwards. Moreover, when scanning the unique identifier after taking a particular soil sample at a particular location position information, e.g., GPS-coordinates or the like, may be assigned to the soil container 52a by aid of the external device 48a, such that enzymatic activity measured from a particular soil sample can be late precisely assigned to the location where the particular soil sample was taken.

Figure 6 shows three schematic diagrams to explain the functionality of the measuring device 12a. The measuring device 12a is configured for measuring at least one enzymatic activity parameter 44a of the at least one type of enzyme in the soil sample during the chemical reaction process. An upper diagram of figure 6 shows how calibration of the detection unit 20a works. On a horizontal axis 80a of the upper diagram a light intensity is plotted in arbitrary units. On a vertical axis 82a a concentration of an optically active reaction product is plotted in arbitrary units. Reference points, such as reference point 84a, are displayed in hollow circles in the upper diagram and represent light intensities at known concentrations of the optically active reaction product, because they are derived from the luminescence of the optically active reaction products contained in the calibration wells 34a, 36a, 38a, 40a, 42a. Measuring points, such as measuring point 86a, are displayed as stars in the upper diagram and represent light intensities of the reaction wells 24a, 26a, 28a, 30a, 32a due to the luminescence of the optically active reaction products released by the chemical reaction process with the enzymes contained in the soil sample reacting with the respective substrates in the reaction wells 24a, 26a, 28a, 30a, 32a. A calibration curve 88a is then fitted by the processing unit 22a through the measuring points.

On a horizontal axis 90a of a lower left diagram of figure 6 a time is plotted in minutes. On a vertical axis 92a of the lower left diagram a light intensity is plotted in arbitrary units. A measuring curve 94a represents the course of the light intensity of one of the reaction wells 24a, 26a, 28a, 30a, 32a over time. During the chemical reaction process between the substrates in the reaction wells 24a, 26a, 28a, 30a, 32a and the enzymes contained in the soil sample the detection unit 20a periodically detects the light intensity of each of the reaction wells 24a, 26a, 28a, 30a, 32a of the reaction element. The measuring curve 94a is determined by the processing unit 22a by extracting data received by the detection unit 20a, for example by extracting RGB values of pictures taken of the reaction element 16a by the detection unit 20a. As can be seen by the course of the measuring curve 94a the light intensity increases over time since the amount of optical active product released by the chemical reaction process increases until the measuring curve 94a approaches a limit value after a certain time has elapsed, because the chemical reaction process comes to a standstill as there are no more enzymes in the soil sample to keep the reaction in progress. The processing unit 22a then determines a slope 96a of the measuring curve 94a as the enzymatic activity parameter 44a, in particular the enzymatic activity rate, of the enzyme which was targeted by a particular substrate in one ore more of the reaction wells 24a, 26a, 28a, 30a, 32a.

On a horizontal axis 98a of a lower right diagram in figure 6 the time is plotted in seconds. On a vertical axis 100a of the lower right diagram a concentration of optically active reaction product, e.g., MUF, is plotted in arbitrary units. Measuring points, such as a measuring point 102a, in the lower right diagram indicate a certain concentration of an optically active reaction product at a certain time and are calculated by the processing unit 22a by comparison of the measuring curve 94a and the calibration curve 88a. Subsequently, the processing unit 22a calculates the amount of a targeted group of enzymes in the soil sample.

Figure 7 shows a schematic flow chart of a method for measuring soil enzymatic activity. The method may be carried out with the measuring system 10a as previously described. The method comprises at least two method steps 118a, 120a. In a first method step 118a of the method at least one reaction element 16a containing at least one substrate embedded in a gel 18a (cf. figure 3) is brought into direct contact with a soil sample to initiate at least one chemical reaction process between at least one type of enzyme in the soil sample and the substrate, wherein at least one optically active reaction product released by the chemical reaction process is detected. In a second method step 120a at least one enzymatic activity parameter 44a (cf. figure 6), in particular an enzymatic rate, of the enzyme in the soil sample is measured directly during the chemical reaction process.

Figures 8 and 9 show two further embodiments of the invention. The following descriptions and the drawings are essentially limited to the differences between the embodiment examples, although reference may in principle also be made to the drawings and/or the description of the other embodiment examples, in particular of figures 1 to 7, with respect to components having the same denomination, in particular with respect to components having the same reference signs. To distinguish the embodiment examples, the letter a is placed after the reference signs of the embodiment example in figures 1 to 7. In the embodiments of figures 8 and 9, the letter a is replaced by the letters b and c.

Figure 8 shows a schematic illustration of a measuring system 10b in another embodiment of the invention.

The measuring system 10b includes at least one measuring device 12b. The measuring device 12b comprises a reaction unit 14b with at least one reaction element (not shown here, cf. reaction element 16a in figure 3) containing at least one substrate (not shown) embedded in a gel (not shown here, cf. gel 18a in figure 3), the reaction element being configured for directly contacting a soil sample (not shown) and enabling at least one chemical reaction process between the substrate and at least one type of enzyme in the soil sample. The measuring device 12b further comprises a detection unit (not shown here, cf. detection unit 20a in figure 2) for detecting at least one optically active reaction product (not shown) released by the chemical reaction process. Furthermore, the measuring device 12b comprises a processing unit (not shown here, cf. processing unit 22a in figure 2) for processing of data detected by means of the detection unit. The measuring device 12b is configured for measuring at least one enzymatic activity parameter (not shown here, cf. enzymatic activity parameter 44a in figure 6) of the at least one type of enzyme in the soil sample during the chemical reaction process.

The measuring system 10b differs from the measuring system 10a of the preceding embodiment essentially with respect to a configuration of a sampling unit 50b.The sampling unit 50b of the measuring system 10b comprises a holding unit 68b for holding a plurality of soil containers 52b, 64b. In figure 8 a soil container 52b and a further soil container 64b of the sampling unit 50b are shown exemplarily. The holding unit 68b includes a rotating drum 70b. The rotating drum 70b is configured for successively bringing different soil samples in each of the soil containers 52b, 64b into contact with at least one reaction element of the reaction unit 14b.

In figure 9 a measuring system 10c in another embodiment of the invention is shown. As in the previous embodiments the measuring system 10c includes at least one measuring device 12c. The measuring device 12c comprises a reaction unit 14c with at least one reaction element 16c containing at least one substrate (not shown) embedded in a gel (not shown here, cf. gel 18a in figure 3), the reaction element 16c being configured for directly contacting a soil sample (not shown) and enabling at least one chemical reaction process between the substrate and at least one type of enzyme in the soil sample. The measuring device 12c further comprises a detection unit 20c for detecting at least one optically active reaction product released by the chemical reaction process. Furthermore, the measuring device 12c comprises a processing unit (not shown here, cf. processing unit 22a in figure 2) for processing of data detected by means of the detection unit 20c. The measuring device 12c is configured for measuring at least one enzymatic activity parameter (not shown here, cf. enzymatic activity parameter 44a in figure 6) of the at least one type of enzyme in the soil sample during the chemical reaction process.

In contrast to the first embodiment of the measuring system 10a the measuring device 12c in the embodiment of figure 9 is miniaturized and portable. The measuring system 10c comprises at least a further measuring device 74c, which is at least substantially identical to the measuring device 12c and which is stackable with the measuring device 12c. The measuring system 10c comprises a plurality of further measuring devices, which, however, are not all marked with reference signs in figure 9 for ease of illustration.

The measuring system 10c is particularly suitable for carrying out a further development of the method described above with reference to figure 7. In this further development of the method, a plurality of measuring devices 12c, 74c are successively brought into direct contact with soil samples at different locations spaced apart from each other wherein in each soil sample at least one enzymatic activity parameter is measured simultaneously. For example a user of the measuring system 10c may bring the reaction element 16c of the reaction unit 14c of the measuring device 12c into direct contact with a first soil sample at a first location, thereby initiating at least one first reaction process between the substrate contained in the reaction element 16c and at least one type of enzyme in the first soil sample and then can move to a further location bringing a further reaction element (not shown) of the further measuring device into direct contact with a further soil sample, thereby initiating at least one further reaction process between the substrate contained in the further reaction element and at least one type of enzyme in the further soil sample and so on. In each of the measuring devices 12c, 74c at least one enzymatic activity parameter is then measured simultaneously. The measuring devices 12c, 74c can then be collected again in reverse order after the measurements are finished and may be used again for further measurements by refreshing the respective reaction elements 16c with new substrates embedded in the gel or by replacing them with new ones. Reference numerals

10 measuring system

12 measuring device

14 reaction unit

16 reaction element

18 gel

20 detection unit

22 processing unit

24 reaction well

26 reaction well

28 reaction well

30 reaction well

32 reaction well

34 calibration well

36 calibration well

38 calibration well

40 calibration well

42 calibration well

44 enzymatic activity parameter

46 unique identifier

48 external device

50 sampling unit

52 soil container

54 first fixing element

56 second fixing element

58 sieve

60 first cooperating fixing element

62 second cooperating fixing element

64 further soil container

66 unique identifier holding unit rotating drum communication unit further measuring device first cooperating fixing element second cooperating fixing element horizontal axis vertical axis reference point measuring point calibration curve horizontal axis verticals axis measuring curve slope horizontal axis vertical axis measuring point housing display unit drawer holder lighting element light sensor element first method step second method step