GB2531622A - Method and device for measuring enzymatic activity of polysaccharide-hydrolying enzymes - Google Patents

Abstract

This invention relates to a method for detecting and/or measuring the activity of a polysaccharide-hydrolysing enzyme (such as an amylase, cellulase, diastase, glucanase, xylanase or NSP). The method includes contacting a sample suspected of containing such an enzyme with a complex of a polysaccharide that is hydrolysable by the enzyme (such as starch, glycogen, arabinoxylan, cellulose, chitin or pectin) and an electrically active signal species or progenitor (such as Phadebas, Cibacron Brilliant Blue FN-G, ferricyanide, ferrocyanide, methylene blue, silver nano-particles or triiodide ions) such that action of the enzyme on the complex causes the release of the signal species or progenitor and the signal species is detected by means of a change in an electrical property (such as potential, capacitance, current, resistance, impedance, phase). Also disclosed is a device suitable for carrying out this method.

Description

METHOD AND DEVICE FOR MEASURING ENZYMATIC ACTIVITY OF POLYSACCHARIDE-HYDROLYSING ENZYMES

TECHNICAL FIELD

The present invention relates to a method for measuring the enzymatic activity of polysaccharide-hydrolysing enzymes. The invention also relates to a device for measuring said enzymatic activity.

BACKGROUND

Enzymatic degradation of polysaccharides into their smaller subunits (usually tri, di and mono-saccharides) is utilised in a number of industrial processes (e.g. food, drink and feed production and paper/pulp technology) and is a key component of the metabolic process in living organisms (e.g. degradation of starch to maltose and glucose units). It is therefore highly desirable to be able to monitor the activity and presence of polysaccharide-hydrolysing enzymes that provide for enzymatic degradation. The monitoring of the presence of such enzymes may also provide valuable information about a sample, for example, in pathology or forensics. The monitoring of the enzyme activity may also be indicative of the activity of organisms in the environment.

Therefore, it is desirable to have a viable assay and device to determine the activity of such enzymes in order to monitor their presence in a sample, their activity in a process and/or as an indication of metabolic function and, thereby, health of an organism or a person.

In human health, amylase activity in saliva has been shown to be an important marker of stress. Starches (amylases), cellulose (glucanases and cellobiase) and xylanose (xylanases) are three common examples of polysaccharides and their concomitant degradative enzymes. Generally, assays of enzymatic activity have been devoted to either non-starch polysaccharide hydrolysing enzymes (NSPs; cellulases and xylanases) or starch-based hydrolysing enzymes (amylases).

There are a number of known assay strategies available for the determination of the activity of non-starch-based degradative enzymes as well as of the amylases. For example, assays to determine the enzymatic activity of starch-degrading enzymes have historically focused on alpha amylase (EC 3.2.1.1) due to its central role in the hydrolysis of starch. Several laboratory-based kits for assaying alpha amylase activity are commercially available, such an: Amylase Activity Colorimetric Assay Kit available from BioVision, Inc; an Alpha Amylase Salivary Kinetic Reaction Kit available from Salimetrics; and Amylase Test Products available from Magle. These kits suffer from high cost per sample and inaccessibility for the general public (both due to personnel and infrastructure requirements) and analysis time (at least 30 minutes).

Several proposals have been made for rapid, easy-to-use diagnostic devices to determine the alpha amylase activity in a sample or a person.

One proposal utilises an arrangement of three enzymes and an electron mediator for an amperometric detection of alpha amylase activity (Mahosenaho, M.; Caprio, F.; Micheli, L.; Sesay, A. M.; Palleschi, G.; Virtanen, V. Microchimica Acta 2010, 170, 243). Another proposal utilises an antibody for molecular recognition and amperometric detection of amylase and levels thereof (Aluoch, A. 0.; Sadik, 0. A.; Bedi, G. Anal. Biochem. 2005, 340, 136).

Yet another proposal uses photometric detection of a chromophore that is liberated from a modified oligosaccharide by the action of the alpha amylase present in the sample (Yamaguchi, M.; Toyama University, Nipro Corporation, Yamaha Hatsudoki Kabushiki Kaisha: USA, 2007 US 7186696 B2, US 7183069, US 20050124060 Al).

WO 2009/017188A (MORITA, Mitsuhiro; (JP), BABA, Toshiaki; (JP).,YOSHIDA, Hiroshi; (JP).,NISHIMURA, Emi; (JP) discloses a similar method to Nipro above, but with electrochemical detection that uses an immobilised enzyme, electron mediator and polysaccharide to generate an amperometric response that correlates to the alpha amylase activity.

Thus, in the prior art there are provided many examples of diagnostic devices and methods to determine, for example, the alpha amylase activity in a sample or a person. Some of the problems associated with these devices and methods are that: i) glucose that is present in a sample will cause a reading that is higher than the actual activity; fi) the enzyme activity is not actually measured; and iii) the cost of the reagents to create a sensor is relatively high.

Although said prior art devices and methods to some extent may alleviate the problems of easy-to-use diagnostic devices to determine the alpha amylase activity in a sample or a person there is still a need for further improvements of devices and related methods so as to provide a device and method that are easy to handle and cost-effective, while providing the possibility of improved detection and measurements of an enzymatic activity of polysaccharide-hydrolysing enzymes such as alpha amylase.

SUMMARY

In view of known test arrangements, it is an objective to provide an improved or alternative method for measuring an enzymatic activity of a polysaccharide-hydrolysing enzyme.

The objective is wholly or partially achieved by a method and device disclosed herein.

The method that has been developed avoids interference from glucose, a limitation of previous electrochemical methods. The enzyme activity is directly correlated to the analytical signal that is read. A device using the method can be inexpensive to produce and easy to handle.

The method relies on detecting an electrical signal at a set of electrodes due to the polysaccharide-hydrolysing enzyme's activity that causes the release of an electro-active substance from a polysaccharide/electro-active species complex. This electrical signal is related to the activity of the polysaccharide-hydrolysing enzyme in the sample presented to an active area of a biosensor comprising the electrodes. In this way, there is provided a simple and rapid method for measuring activity of polysaccharide-hydrolysing enzyme(s). A rapid and disposable diagnostic device that may be used in the method is also presented.

In one aspect the invention provides a method for detecting and/or measuring a polysaccharide-hydrolysing activity of an enzyme and/or quantifying the amount of the enzyme in a sample, comprising: (a) providing a complex of a polysaccharide that is hydrolysable by the enzyme and a signal species such that action of the enzyme on the complex causes liberation of the signal species; (b) contacting a sample suspected to contain a said enzyme with said complex; and (c) detecting signal species liberated from the complex.

The enzyme may be an amylase.

The signal species is desirably an electro-active species. The detection step may then employ measurement of an electrical parameter that is affected by the liberated signal species.

In the complex, the signal species may be chemically bound to the polysaccharide and/or physically bound, e.g. being trapped in a matrix.

The signal species may be detectable visually or colorimetrically or spectroscopically, preferably in addition to being electro-active. Thus it may be detected using a simple colorimeter (which may be constituted by a mobile phone with a suitable app).

Whereas the signal species is normally detectable itself, it may be a substance that takes part in a reaction or series of reactions which produces a product that is detected.

A portion comprising the complex may contain a controlled amount of an enzyme inhibitor. Thus no liberation of signal species will be detected unless the amount of enzyme in the sample exceeds the amount that can be inhibited by the inhibitor. Thus any detection of the signal species indicates that the enzyme activity exceeds a threshold value. There may be a multiplicity of complex portions containing respective different amounts of the inhibitor. Then determining which portions do lead to detection of liberated signal species, and which do not, provides a simple indication that the enzyme activity is within a particular range.

In a second aspect the invention provides a device for carrying out such a method. In a preferred type of embodiment this comprises a portion carrying a complex of a polysaccharide and an electro-active species and a portion arranged with an electrical detection member. The device may comprise: a. a solid, water-insoluble, electrically insulating support substrate (either porous or non-porous); b. water-insoluble conducting materials forming electrodes affixed to the aforementioned substrate via adsorption, immobilisation and/or intermolecular interactions such that the conducting material remains affixed to the substrate during the assay; c. complex of a polysaccharide and an electro-active species, which is adsorbed on the support and/or on top of one or more of the electrodes.

Preferably the electro-active species is also optically detectable.

A device may include an inhibitor; so that enzymatic activity may be quantified by use of an inhibitor or series of inhibitors. Such a device may comprise an elongated element having three portions along a longitudinal direction thereof: a first portion arranged at a longitudinal end that comprises a complex; a second portion arranged at an opposite longitudinal end that forms a sample application portions; and a third portion arranged between the first and second portion, the third portion comprising an inhibitor of polysaccharide hydrolysis.

In a further aspect the invention provides a kit of parts for measuring an enzymatic activity of a polysaccharide-hydrolysing enzyme comprising a device, reagents, sample collector, electrical reader, display, recording method, and a power source or means for connection to a power source.

An embodiment of the invention may provide means to measure the activity of an enzyme that may be a biological indicator for a disease, condition, treatment effect, psychological state or other relevant biological state. It may also provide a measurement of activity of an enzyme that may be used for biological based processes in industry or medicine.

The first step a) comprises the provision of a complex of a polysaccharide and an active species (or signal species). This complex may have low solubility in water and therefore the active species is not accessible for detection. One example of such a complex is the commercially available complex Phadebas (available from Magle Life Sciences, USA/Canada, 155 Brookline Street #5, Cambridge, MA 02139, USA). It is a water-insoluble, cross-linked starch polymer carrying a blue dye (Cibacron brilliant blue FN-G) as the active species. The complex forms a globular microsphere of defined size. The microsphere is insoluble in water and the blue dye is covalently bound to it. The blue dye as such is water soluble and can be visibly be detected or detected and quantified in a spectrophotometer at a wavelength of 620 nm.

It has now also been found that the blue dye of Phadebas may also serve as an electro-active species. Thus it may be detected and quantified based on its electroactive properties, wherein an electrical signal is eventually formed upon an enzymatic release of the blue dye from the complex. For example, this may be achieved by the use of a potentiostat as is further explained below.

Other ways of electrically detecting the active species may rely on electrical signals that may be capacitance, current, potential, resistance, impedance, phase or combination of electrical signals listed above. The electrical detection may be from a separate reader the device is inserted into or placed on, or an integral part of an electronic circuit with reader, electronics, display, power source, or wireless communication on a substrate that may be printed.

A person skilled in the art appreciates that there are many possible active species that may be linked to polysaccharides for use in the present method. Examples of such electro-active species are Cibacron Brilliant Blue FN-G, ferri/ferro-cyanide, methylene blue and silver nano-particles. Any electroactive or coloured moiety that is complexed or buried inside a polysaccharide may also be used.

In a polysaccharide hydrolysing step, a sample that may contain a polysaccharide-hydrolysing enzyme such as amylase may be contacted with the complex for a short period of time (seconds to minutes) at room temperature.

The method may further comprise an inhibition step. In the inhibition step, an inhibitor of the enzymatic release of the active species is provided. The inhibitor may be a substance binding to the enzyme as such or the polysaccharide as such or have any other mode of action that prevents the enzymatic release of the active species from the complex. An example of such a substance is HgCl2 that blocks the enzymatic hydrolysis action of amylase.

Further, examples of inhibitors are luteolin, scutellarein, tris(hydroxymethyl)aminomethane, antibodies, molecular imprinted polymers (MIPs), peptides, aptamers, and any other small or large molecule that specifically inhibits the enzymatic activity of the polysaccharide-hydrolysing enzyme.

In a detection step, the released active species is detected and may be quantified. Purely as an example, a released Phadebas blue dye dissolves in a water solution of the sample and colours the solution blue. This may easily be detected and correlated to the amount of inhibitor added. In a complete inhibition of the blue dye release by the inhibitor, the water solution does not contain any blue dye dissolved therein. Thus, the activity of the enzyme may easily be quantified by an inhibitor titration, for example. The blue dye may alternatively or additionally be detected using its electro-active properties, thereby providing an improved method providing accurate measurements that are easy to perform.

The method described herein is also useful in any application where qualitative or quantitative knowledge of enzyme activity is desired, such as clinical laboratory assays with associated equipment and devices, industrial laboratory assays with associated equipment and devices, clinical, industrial, or environmental field work, or other related applications.

ADVANTAGES: A device using electrical measurement can have the following advantages: the simplicity of the electronics required, inexpensive fabrication, an accurate quantitative assay, a device which is small and portable, a device which is easy to use (user-friendly) and applicable to point-of-care (POC) use.

The electro-active species or substance may be a species that can take part in an electrode reaction and/or that can be adsorbed on an electrode to affect an electrical property thereof. In particular, it may be a substance that undergoes a change of oxidation state, or the breaking or formation of chemical bonds, in a charge-transfer step. Examples of such electro-active species are Cibacron Brilliant Blue FN-G, ferri/ferrocyanide, methylene blue, and silver nano-particles. Many electro-active species are also substances that may be detected by their colour, and therefore also be useful in a colorimetric method.

An electro-active species can lead to an electric signal, namely a signal generated and/or transferred by electronic means, in particular, a signal that may be transferred by use of electricity. Some ways in which an electro-active species may generate a detectable signal when interacting with an electrode include, but are not limited to, by affecting the electrical resistance/impedance of a circuit, by inducing or changing a current flow within a circuit, or by changing the potential or by generating a current in a circuit by either losing or gaining electrons.

A complex of a polysaccharide and an electro-active species is here meant to include the two components of said active species and said polysaccharide that are joined together by, for example, a chemical bond so as to form a complex. The active species may be associated with the polysaccharide entity via a multitude of mechanisms. These may be, but are not limited to, covalent attachment, entrapment, coulombic attraction, and/or a combination of intermolecular interactions.

The complex may be provided on a support, namely a solid, water-insoluble, electrically insulating substrate that may comprise various porous and/or non-porous materials that have varying degrees of flexibility (e.g. paper, thermoplastics such as polystyrene, polytetrafluoroethylene, silicon dioxide, silicon, aluminum oxide, ceramic, glass, or any other material having a surface suitable for supporting the complex and possibly other reagents and components such as electrodes.

By a biosensing device is here meant a self-contained sensor capable of detecting the presence of a biological entity or activity.

Some embodiments of the invention will now be described in more detail with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A is a top plan view of Embodiment A: a lateral flow device incorporating an electrode, a polysaccharide activity inhibitor(s), and a polysaccharide/electro-active moiety complex.

Figure 1B is a section on B-B in Fig 1A.

Figures 2A and 2B are similar views of Embodiment B: an electrode with a polysaccharide/electro-active moiety complex adsorbed on top of the electrode.

Figure 3 is a longitudinal section through Embodiment C: an optically-based lateral-flow device utilising enzyme inhibitor(s).

Figure 4 is a top plan view of Embodiment D: a linear design for digitised enzyme activity quantitation utilising multiple components of Embodiment C. Figure 5 is a top plan view of Embodiment E: a radial design for digitised enzyme activity quantitation utilising multiple components of Embodiment C. Figure 6 is a graph of signal current versus time which represents a chronoamperometric signal for a Phadebas-modified screen-printed carbon electrode (SPCE) with human salivary a-amylase (HsAA) applied.

Figure 7 shows a low activity calibration curve at 60 seconds after sample application.

Figure 8 shows a high activity calibration curve at 180 seconds after sample application.

DETAILED DESCRIPTION OF EMBODIMENTS

Physical description of Embodiment A (Figures 1A and 1B) Embodiment A is a lateral flow device incorporating an electrode, alpha-amylase activity inhibitor(s), and a polysaccharide/electro-active moiety complex.

An insulating substrate 10 comprises or is modified with a liquid wicking or fluid flow conducting mechanism (such as a wicking material or microfluidic channel filled either by capillary action or by some form of active or passive pump). Adjacent one end, there is an electrical connector zone 12 from which electrodes 14 extend some way along the substrate. A polysaccharide/electro-active moiety complex (the complex) (Phadebas) is adsorbed, immobilised or otherwise held in place in the liquid wicking substance or conducting mechanism near the Connector Zone end of the substrate but such that the Connector Zone is free of the complex. A mixture containing alpha-amylase inhibitor(s) is adsorbed/absorbed, immobilised or otherwise held onto/into the liquid conducting mechanism at the opposite end of the substrate relative to the complex position. An electrically conducting material extends from the Connector Zone end of the substrate such that the electrodes that are formed by the electrically conducting material cover the complex. A means for at least two of the electrodes to be connected to a power source/signal detection device is provided; 2) the end of the substrate opposite the Connector Zone end and past the inhibitor material is coated with an insulating layer such that a Sample Application Zone is formed.

Operational description of Embodiment A

The sensor is connected electrically to a power source/signal detection device via the Connector Zone and a potential is applied to the electrodes. Sample is applied directly to the Sample Application Zone and travels to the polysaccharide/electroactive moiety complex through the liquid transport system. During this translocation process the sample encounters the inhibitor(s), allowing for a reaction to occur.

Upon reaching the polysaccharide/electro-active moiety complex, uninhibited alpha-amylase degrades the polysaccharide/electro-active moiety complex via enzymatic action. This enzymatic degradation of the complex results in the release of the electro-active moiety. Upon release from the polysaccharide/electro-active moiety complex, the electro-active moiety moves to the electrode, is reduced or oxidised at the electrode and thereby produces a signal proportional to the uninhibited enzymatic activity. An array of sensors, each containing a different amount of inhibitor, can be used to quantitate the alpha-amylase level/activity in the sample.

Physical description of Embodiment B (Figure 2):

The sensor as illustrated in figure 2 comprises three components, namely: 1) a solid, water-insoluble, electrically insulating support substrate (either porous or non-porous); 2) water-insoluble conducting materials (electrodes) affixed to the aforementioned substrate via adsorption, immobilisation and/or intermolecular interactions such that the conducting material remains affixed to the substrate for the period of the assay; and 3) a polysaccharide/electro-active and/or optically detectable complex which is adsorbed on top of one or more of the electrodes.

The polysaccharide may be starch or non-starch. It may be a substrate for a specific polysaccharide-hydrolysing enzyme or a general substrate for a variety of polysaccharide-hydrolysing enzymes.

The solid, water-insoluble, electrically insulating support substrate may comprise various porous and/or non-porous materials that have varying degrees of flexibility and are electrically insulating (e.g. paper, polystyrene, polytetrafluoroethylene, thermoplastic, silicon dioxide, silicon, aluminum oxide, ceramic, or glass). The electrodes will form conducting tracks that are independently addressable. These electrodes may be incorporated within and/or exist on the surface of the support. The electrodes may be composed of any conducting substance (carbon, carbon nano-tubes (CNTs), platinum, silver, gold, rhodium, aluminum, conductive polymers such as poly(3,4-ethylenedioxythiophene) and polypyrrole), combination of one or more conducting and/or non-conducting substances and be applied in appropriate patterns to the substrate in a variety of manners (e.g. screen printing, contact printing, roll-to-roll, vapour deposition followed by laser ablation, ink jet, thermal, or other). The electro-active specie(s) may be any moiety that affects an electrical circuit such that a detectable signal is generated when it is free to interact with the electrodes of the electrical circuit. Some ways in which an electro-active species may generate a detectable signal when interacting with an electrode are by affecting the electrical resistance/impedance for the circuit, by inducing or changing a current flow within the circuit, by changing the potential or by generating a current in the circuit by either losing or gaining electrons. The electro-active species may be associated with the polysaccharide entity via a multitude of mechanisms. These include covalent attachment, entrapment, coulombic attraction, and/or a combination of intermolecular interactions. The electro-active species may be a dielectric molecule, an easily polarisable molecule, a molecule that can easily gain and/or lose electrons, and/or a zwitterion; it also may be amphiphilic or water soluble.

Various formats for the device may be provided. The device could, for example be configured as a single or multiple analysis disposable analysis strip that may include power, read-out and other integrated electronic components, a single or multi-analysis strip to be coupled with a reader device, or as a reusable device.

The device may also be a screen-printed electrode with a polysaccharide/electroactive moiety complex adsorbed on top of the electrode. A schematic view of such an electrode is shown in Fig. 2. (Embodiment A may be generally similar.) The device is configured in a linear fashion with reactants placed strategically in line. In a variant, the device is configured in a radial fashion from 1 degree to 360 degrees around a central point with reactants placed strategically around or out from the central point. The radial device may include several radii or spirals from a central point.

The device may include one or several components that make analysis for an application possible without need for external equipment which may include: the device, reagents, sample collector, electrical reader, display, recording method, power source or connection to power source, or other ancillary supplies.

As in Embodiment A, there is a Connector Zone which is free of the complex. An electrically insulating layer covers the device with the following exceptions: 1) a segment of the end of the biosensor that does not include the complex remains free of the electrically insulating top layer such that the electrodes are exposed for connection to a power source/signal detection device; 2) the central area of the complex such that sample may be applied to this area. Electrodes of the biosensor are connected to the power source/signal detection device through the Connector Zone. An insulating material covers the electrodes and polysaccharide/electro-active moiety complex except at the Connector Zone and Sample Application Zone.

Operational description of Embodiment B:

A potential is applied to a working electrode by either connecting the electrodes to a power source/signal detection device via the Connector Zone or by otherwise generating a potential difference between the electrodes. Sample is applied directly to the Sample Application Zone. Enzymatic action of the sample degrades the polysaccharide/electro-active moiety complex, resulting in the release of the electroactive moiety. Upon release from the polysaccharide/electro-active moiety complex, the electro-active moiety diffuses to the electrode, is reduced or oxidised at the electrode and thereby produces a signal proportional to the enzymatic activity.

The electro-active species is unable to interact with the electrodes while associated with the polysaccharide molecule, even in the presence of water and, therefore, unable to produce an electrical signal. Only upon enzymatic activity of the hydrolysing enzyme on the polysaccharide, does the electro-active species become available to interact with the electrode(s) and thereby produce an electrical signal that is correlated to the enzymatic activity of the applied sample.

In a specific example, using the two-electrode, screen-printed carbon electrode (SPCE) illustrated in figure 2, we employed a Phadebas suspension of 0.352 grams per milliliter of Phadebas beads (beads used to coat indicator paper (Phadebas Forensic) for detection of amylase, sample from Magle AB, Sweden) in 0.10 M HEPES buffer, pH 7.0; and Human Salivary Alpha Amylase (HsAA; product no: 120- 17, Lee Biosolutions, USA), diluted in 10 mM Tris HCI, pH 7.5 to activity between 1.67 and 1715 Units per milliliter (U/mL).

Procedure: 10 microliters of the Phadebas suspension (0.0352 g Phadebas beads suspended in 1.0 mL o f0.1 M HEPES aqueous buffer, pH 7.0) was applied to the working area of the screen-printed carbon electrode (SPCE) and allowed to dry at room temperature. This modified SPCE was attached to a potentiostat (PalmSens BV Emstat, The Netherlands) which was configured in the Chronoamperometry mode to apply +0.8 Volts for 400 seconds. 40 yL of the HsAA solution was deposited on the working area of the modified SPCE, the potentiostat was initiated and the corresponding current was recorded.

Results: Upon application of the enzyme-containing solution to the working area of the modified SPCE and application of the +0.8 volts, a current was generated and recorded by the potentiostat (see below). The current is due to oxidation of the electroactive dye molecules that are released from the Phadebas beads due to the enzymatic activity of the alpha amylase-containing sample. More specifically, when an aqueous solution containing alpha amylase enzyme is applied to the Phadebasmodified area of the SPCE, digestion of the starch-based encapsulating material by the alpha amylase commences. Alpha amylase cleaves the a(1,4) glycosidic linkages between glucose molecules in starch. This digestive process produces water-soluble glucose and maltose units; thus, liberating the electroactive dye and allowing it to diffuse to the electrode surface where it can be oxidized by the applied potential.

Fig. 6 shows the current response from the biosensor at a particular point in time, plotted against the HsAA activity for a low HsAA activity range (1.67 U/mL to 107.2 U/mL) and a high HsAA activity range (107.2 U/mL to 1715 U/mL). The low activity range calibration curve showed the best fit to a linear trend line (R2 = 0.98) at the 60 second current reading while the high activity range calibration curve showed the best fit to a linear trend line (R2 = 0.93) at the 180 second current reading seen in Figures 7 and 8.

This demonstrates that the method can be applied to measure a biologically relevant concentration of enzyme, and this can be quantitated using a pre-determined calibration by electrochemical detection of a released electroactive moiety from the complex.

Physical description of Embodiment C (Figure 3):

Fig. 3 is a sectional view of a paper strip without or with a transparent polymer coating. It has an interaction zone which may contain (soluble) inhibitors. A colour reagent zone may contain suitable molecules/structures modified by the enzyme.

The threshold for the colour reaction is set by the concentration of the inhibitor.

Colour changes for sufficiently large enzyme activity may increase with time.

Operational description of Embodiment C:

Sample is placed on the uncovered area of the untreated paper. Capillary action draws the sample through the interaction zone where the enzyme may interact with inhibitors.

Upon reaching the colour reagent zone, uninhibited alpha-amylase degrades the polysaccharide/dye complex via enzymatic action. This enzymatic degradation of the complex results in the release of the coloured species. Release of the coloured species from the complex allows for visual and/or instrumental detection of the coloured species in a quantitation manner. Increasing amounts of enzyme activity results in increased colour density.

A multiplicity of such devices with different amounts of inhibitor can be used to digitise the enzyme actively level by means of inhibition of the enzymatic activity. A radial array of this type is shown in Fig 5. This technique could be used in electrochemical and/or colorimetric detection procedures, wherein an electrochemical procedure may provide more accurate and simple measurements. In addition, the digitisation could be simple yes/no, a semi-quantitative or a fully quantitative result. In the present example, the inhibitor was HgCl2, and we used a colorimetric detection procedure. Nonetheless, the skilled person will in view of the present disclosure appreciate that the colorimetric detection procedure could be replaced with an electrochemical detection procedure as illustrated in the earlier examples.

Claims (18)

1. CLAIMS: 1. A method for detecting and/or measuring a polysaccharide-hydrolysing activity of an enzyme and/or quantifying the amount of the enzyme in a sample, comprising: (a) providing a complex of a polysaccharide that is hydrolysable by the enzyme and an electrically active signal species or progenitor thereof such that action of the enzyme on the complex causes liberation of the signal species or progenitor; (b) contacting a sample suspected to contain a said enzyme with said complex; and (c) detecting signal species liberated from the complex, or generated by means of a progenitor liberated from the complex, by means of an electrical property thereof.
2. The method of claim 1 wherein the enzyme is an amylase.
3. 3. The method of claim 1 or claim 2 wherein the detection step employs measurement of an electrical parameter that is affected by the signal species.
4. 4. The method of any preceding claim wherein, in the complex, the signal species or progenitor is chemically bound to the polysaccharide.
5. 5. The method of any preceding claim wherein, in the complex, the signal species or progenitor is physically bound to the polysaccharide.
6. 6. The method of any preceding claim wherein the signal species is detectable visually or colorimetrically or spectroscopically, in addition to being electro-active.
7. 7. A method according to any preceding claim wherein said sample is contacted with a controlled amount of an enzyme inhibitor, so that no signal species will be detected unless the amount of enzyme in the sample exceeds the amount that can be inhibited by the inhibitor.
8. 8. A method according to claim 7 wherein respective portions of the sample are contacted with respective different amounts of the inhibitor so that determining which portions do lead to detection of signal species, and which do not, indicates that the enzyme activity is within a particular range.
9. 9. A method according to any preceding claim wherein the complex contains bound signal species.
10. 10. A method according to any of claims 1 to 8 wherein the complex contains bound progenitor and the method includes a step of using liberated progenitor to produce the signal species.
11. 11. A method for detecting and/or measuring an activity of and/or quantifying an enzyme in a sample substantially as described and exemplified herein.
12. 12. A device for carrying out a method according to any preceding claim comprising a substrate and means for use in electrical detection comprising electrodes which extend on the substrate; said complex of a polysaccharide and an electro-active species or progenitor thereof being provided on the substrate; there being a sample application zone, the arrangement being such that a sample applied at the application zone can contact the complex, whereafter liberated signal species or precursor can move to contact an electrode, a precursor, if necessary, undergoing reaction(s) to produce a detectable signal species.
13. 13. A device according to claim 12 wherein the substrate is a solid, water-insoluble, electrically insulating support element; the electrodes are constituted by water-insoluble conducting materials affixed to the substrate via adsorption, immobilisation and/or intermolecular interactions such that the conducting material remains affixed to the substrate during the assay; and said complex is adsorbed on the support and/or on top of one or more of the electrodes.
14. 14. A device according to claim 12 or claim 13 which comprises an elongate element having three portions along a longitudinal direction thereof a first portion arranged at a longitudinal end that includes said complex; a second portion arranged at an opposite longitudinal end that forms said sample application zone; and a third portion arranged between the first and second portion, the third portion including an inhibitor of polysaccharide hydrolysis.
15. 15. A device according to any of claims 12 to 14 that is adapted to be coupled to a separate reader so that the reader is in electrical communication with said electrodes.
16. 16. A device according to any of claims 12 to 14 that is a stand-alone device, including an integrally formed reader which comprises circuitry, a power source and a display and/or means for wireless communication.
17. 17. A device according to claim 16 wherein the circuitry is printed.
18. 18. A device for carrying out a method for detecting and/or measuring a polysaccharide-hydrolysing activity of an enzyme and/or quantifying the amount of the enzyme in a sample substantially as described herein with reference to and as illustrated in the accompanying drawings.