Method of detecting bacterial infection in a biological sample

Abstract

A method of indicating the presence of a bacterial infection in a biological sample is provided. The method detects a marker for infection by providing a device, the device including a biosensor, an interaction arising between the biosensor and the marker when the marker is present in the biological sample. Contacting at least a part of the biological sample with the biosensor of the device, therefore, provides analysis of the biological sample with respect to the marker by detecting for the interaction between the biosensor and the marker. A preferred marker is the enzyme amylase.

Claims

1. A method of indicating the presence of a bacterial infection in a biological sample obtained from a wound of a subject, other than a pancreatic wound or oral digestive wound, the method comprising: a. providing a device comprising a biosensor, wherein the biosensor includes an α-amylase specific antibody and an interaction arises between the α-amylase specific antibody and α-amylase when the α-amylase is present in the biological sample; b. contacting at least a part of the biological sample with the biosensor of the device; c. analysing the at least a part of the biological sample with respect to the α-amylase by detecting for the interaction between the α-amylase specific antibody and the α-amylase; d. indicating an amount of the α-amylase present in the biological sample using the device; e. repeating steps b.-d. on a blood, plasma, or serum sample obtained from the subject, thereby indicating an amount of α-amylase present in the blood, plasma, or serum sample; and f. comparing the amount of α-amylase in the biological sample with the amount of α-amylase in the blood, plasma, or serum sample, and where the difference in the amount of α-amylase in the biological sample compared with that in the blood, plasma, or serum sample is elevated above 140 IU/L, concluding the elevated amount of α-amylase is indicative of a bacterial infection present in the biological sample.

2. The method according to claim 1, wherein the method is a method of diagnosing infection.

3. The method according to claim 1, wherein the method is a method of quantifying the amount of α-amylase in the at least a part of the biological sample and the blood, plasma, or serum sample.

4. The method according to claim 1, wherein the method is a method of quantifying the concentration of α-amylase and/or the activity of α-amylase in the biological sample relative to the concentration and/or activity of α-amylase in the blood, plasma, or serum sample.

5. The method according to claim 1, wherein the wound is one or more of: a breast, abdomen, buttocks, lower limb, or upper limb wound.

6. The method according to claim 1, wherein the device further comprises one or more or all of: a power source; control electronics; a computer processor; computer memory; a user interface; a user viewable display; a signal output connection; and a signal input connection.

7. The method according to claim 1, wherein the biosensor is: screen printed on to a part of the device; and/or is a graphene based sensor; and/or includes electropolymerization of a component of the biosensor.

8. The method according to claim 1, wherein the biosensor comprises an electrochemical impedance spectroscopy sensor.

9. The method according to of claim 1, wherein the biosensor has a linear response against α-amylase concentration and/or detects concentrations of α-amylase as low as 100 International Units/L (IU/L).

10. The method according to claim 1, wherein the biosensor detects concentrations of α-amylase as low as 1 International Units/L (IU/L).

11. The method according to claim 1, wherein the biosensor has a limit of detection as low as 0.025 U/L.

12. The method according to claim 1, wherein the subject is treated for infection when the amount of α-amylase is elevated in the biological sample compared with that in the blood, plasma, or serum sample.

13. The method according to claim 1, wherein the subject is not treated for infection when the amount of α-amylase is not elevated in the biological sample compared with that in the blood, plasma, or serum sample.

14. The method according to claim 1, wherein the biological sample is a fluid sample obtained from the wound.

15. The method of claim 1, wherein the wound is a torso or limb wound.

16. The method of claim 1, wherein the wound is not contaminated with salivary amylase.

17. The method of claim 1, where the amount of α-amylase in the biological sample is at least 400 IU/L.

18. A method of indicating the presence of a bacterial infection in a biological sample obtained from a wound of a subject, wherein the wound is not associated with a known pancreatic, salivary, or macroamylasemia pattern of amylase distribution, the method comprising: a. providing a device comprising a biosensor, wherein the biosensor includes an α-amylase specific antibody and an interaction arises between the α-amylase specific antibody and α-amylase when the α-amylase is present in the biological sample; b. contacting at least a part of the biological sample with the biosensor of the device; c. analysing the at least a part of the biological sample with respect to the α-amylase by detecting for the interaction between the α-amylase specific antibody and the α-amylase; d. indicating an amount of the α-amylase present in the biological sample using the device; e. repeating steps b.-d. on a blood, plasma, or serum sample obtained from the subject, thereby indicating an amount of α-amylase present in the blood, plasma, or serum sample; and f. comparing the amount of α-amylase in the biological sample with the amount of α-amylase in the blood, plasma, or serum sample, and where the difference in the amount of α-amylase in the biological sample compared with that in the blood, plasma, or serum sample is elevated above 140 IU/L, concluding the elevated amount of α-amylase is indicative of a bacterial infection present in the biological sample.

19. The method of claim 18, where the amount of α-amylase in the biological sample is at least 400 IU/L.

Description

BRIEF SUMMARY OF THE DRAWINGS

(1) Various embodiments of the invention will now be described, by way of example only and with reference to the accompanying drawings in which:

(2) FIG. 1 shows a distribution graph illustrating that detection according to the invention was useful across different types of bacteria, different types of wound, wound dressing, antibiotics, wound site, and depth;

(3) FIG. 2 illustrates experimental results showing the amylase activity for a series of samples, the samples being characterised into the group type “plasma” or the group type “infected fluid”;

(4) FIG. 3A illustrates the difference in results for the two groups of FIG. 2 using the Mann Whitney matched pairs signed test (P=0.002);

(5) FIG. 3B illustrates the minor differences in results for the groups wound sourced sample and blood sourced sample;

(6) FIG. 3C illustrates the differences in results between the “infected fluid” group of FIG. 2 and a series of control samples;

(7) FIG. 4 shows a Western blot analysis of amylase size distribution showing degradation/cleavage within an infected environment;

(8) FIG. 5 illustrates experimental results showing the concentration difference between infected wound fluid and plasma for the same individual patient across a series of patients;

(9) FIG. 6 illustrates the concentration difference in the results for the two groups using the Mann Whitney matched pairs signed test (P<0.0001);

(10) FIG. 7a illustrates the concentration difference (wound/plasma) for infected cases, showing a highly significant difference;

(11) FIG. 7b illustrates the concentration difference (wound/plasma) for non-infected cases, showing no significant difference;

(12) FIG. 7c illustrates the concentration difference between experimental cases and controls, showing a highly significant difference;

(13) FIG. 8A provides a Nyquist plot showing the effective response of an immunosensor, suitable for use in the present invention, to increasing amylase concentrations;

(14) FIG. 8B shows the resulting EIS calibration curve for the immunosensor of FIG. 6A to increasing amylase concentrations;

(15) FIG. 9 provides an EIS calibration curve for the immunosensor of FIG. 6A to increasing concentrations of amylase in mouse plasma;

(16) FIG. 10 provides a Nyquist plot showing the dynamic range detectable when the sensor was exposed to various human plasma concentrations (9.9-1009.9 U/L);

(17) FIG. 11 is a Roche Cobas test or recovery, illustrating linearity of recovery for amylase in plasma and PBS but not linear recovery in infected wound fluid; and

(18) FIG. 12 is a schematic a illustration of the construction of the graphene sensor, including the electropolymerization of polyaniline to the graphene surface.

DETAILED DESCRIPTION

(19) Amylase

(20) Amylase is a naturally produced enzyme which provides for the breakdown of components in foodstuffs consumed by humans, other animals, together with some plants and bacteria. The enzyme acts by catalysing the hydrolysis of the starches present into sugars. Other enzymes then convert those components to glucose to provide an energy source.

(21) The amylase may be generated by salivary, mammary and lacrimal glands and, in the case of a particular isoform in the pancreas. It occurs naturally in humans, animals, plants, fungi and bacteria. More properly this is α-amylase. α-amylase is also known as 1,4-α-D-glucan glucanohydrolase or glycogenase.

(22) In contrast, β-amylase is naturally produced by bacteria, fungi and plant seeds and so may be present in consumed foodstuffs. β-amylase is also known as 1,4-α-D-glucan maltohydrolase. γ-amylase is primarily found in yeast and fungi, but is also naturally produced in some human tissues, particularly within the intestine. γ-amylase is also known as 1,4-α-D-glucan glucohydrolase.

(23) Amylase is known to be an aid to diagnosis, for instance through test blood serum, urine or peritoneal fluid for hyperamylasemia with a view to informing on acute inflammation of the pancreas.

(24) More recently, amylase, particularly α-amylase, has been used clinically in pancreatic enzyme replacement therapy and other treatments.

(25) Detection and Quantification of Amylase

(26) A variety of sensors or other forms of assay are known which can detect amylase in samples, however, they function by detecting the activity of amylase. They do not operate through the detection of concentration, are not capable of the detection of the concentration levels of amylase typically encountered in medical situations and samples and do not have the necessary sensitivity to detect very low concentration levels of amylase in samples. Furthermore, their response is non-linear in some body fluids, with variation in response to the same activity between body fluids. A sensor offering this combination of features is not available in the prior art.

(27) Activity based detection can be negatively affected by deviation on pH away from the 6.7 to 7.0 range optimal for activity and/or due to temperature and/or due to ionic composition and/or due to the composition of the fluid and/or due to reactions, such as chelation, reducing activity.

(28) In later sections, a sensor is described which allows for the accurate detection of low concentrations of amylase in samples.

(29) Initially, however, a use of such a sensor will be described. The use is in the detection of amylase, more specifically the determination of the concentration of amylase, present in a sample so as to inform on the presence of infection at a site associated with the source of the sample. This is a new pattern of amylase distribution, as compared with the four known patterns; pancreatic, salivary, macroamylasemia and combinations of those three.

(30) The applicant has established that this new pattern of amylase distribution is valid and applicable through the following testing.

(31) A library of infections was compiled to represent a variety of patient sex, infection locations, bacteria identities, dressing type and diagnosis. In the illustrated example of FIG. 1, this was formed of the information from 20 patients. The spread of factors was used to investigate whether the new pattern was applicable to a wide variety of different infections, in terms of organism, site, open and closed wounds, superficial and deep infections and the like.

(32) Samples were collected from infection sites and tested using a sensor described further below. Samples were also collected from plasma simultaneously off each patient and tested in the same manner and using the same sensor. Plasma amylase was used as the comparator. Whilst amylase is present in plasma, the levels are regulated. Infection causes enhanced vascular permeability, therefore, large molecules such as amylase are likely to sequester to the site and not be able to drain away because of distorted lymphatics. Amylase is also likely to be secreted by some bacteria.

(33) FIG. 2 shows the amylase activity detected for each of the samples collected. Some samples were of insufficient volume for testing and are shown as absent data points. Testing was possible for 14 samples. FIG. 2 shows whether these were sample associated with an infection site or plasma. The site being infected was established to be true through alternative testing approaches.

(34) Amylase is normally present in human plasma. A substantial difference between the plasma and the infected fluid sample collected off each of the 20 patients was that amylase was detected in each and every one of the infected fluid samples. Many of the plasma samples showed undetectably low concentrations of amylase.

(35) The results of FIG. 2 suggest a marked difference in the amylase activity between plasma samples, where infection is absent, and infection sites, where infection was present.

(36) FIG. 3A shows the outcome when Wilcoxon matched pairs signed rank testing is applied to the results. A separate between the two sample group types is establish with P=0.002. FIG. 3B shows the amylase activity comparison between wound sourced control samples and blood sourced control samples (p=0.3846). FIG. 3C shows the amylase activity comparison between patients providing infected samples and patients providing non-infected samples (p<0.0001, n=20). In FIGS. 2, 3A, 3B and 3C an activity difference is under consideration.

(37) FIG. 4 shows the results from Western blot analysis of amylase size (kDa) distribution from a variety of samples and is indicative of cleavage/degradation of the amylase occurring.

(38) FIG. 5 considers the concentration difference for pairs of results, one of the pair being a plasma sample and the other being an infected site sample taken from the same patient. 20 sample pairs were considered in this case consistent with the patients in the library of FIG. 1. FIG. 5 shows that the difference in concentration between the two samples from the same patient is significant in each and every case tested; 100% increase in concentration in the infected sample in many cases. The sensor showed excellent sensitivity (98%) and high specificity (90%) with respect to infection in the measurement results generated. Sensitivity being the ability to correctly identify those with the infection (true positive rate); specificity being the ability to test correctly to identify those without the infection (true negative rate).

(39) In FIG. 6 the outcome from the application of Mann Whitney matched-pairs signed rank testing is seen. The separation between the two sample types is clearly established with P<0.0001.

(40) FIGS. 7a, 7b and 7c show the outcome from the application of Mann Whitney matched pair signed rank testing. With FIG. 7a being infected cases, FIG. 7b being non-infected cases and FIG. 7c controls.

(41) The results show that the difference in amylase between a site of interest and intravascular fluid (defined above) can be used to determine the presence of infection, or its likelihood, at the site of interest. If the activity difference (site of interest versus intravascular fluid) is considered, the distinction is statistically significant. If the concentration is considered, then the distinction is highly statistically significant. The amylase difference between infected and non-infected sites is highly sensitive to the presence of infection.

(42) The experimental results show that the detection of activity and more usefully concentration difference reveals whether or not infection is present at the site. This information could be used to direct subsequent and further investigations. For instance, a determination as to the nature of the infection could be sought, for instance by microbial culture, microscopy, biochemical testing, molecular testing, immunoglobulin binding to amylase or others. This information on the infection can be combined with information on the incident or procedure leading to the wound and then used by a medical practitioner to diagnose a medical condition and the form of treatment appropriate.

(43) A significant benefit of being able to establish that infection is present at the wound site and needs treatment is that no treatment need be given where no infection is established to be present. At the moment, clinical practice errs on the side of caution and so antibiotics, as infection treatment, are administered in most situations. This represents unnecessary antibiotic use and so contributes to wasted costs and, more significantly, increased build-up of resistance to antibiotics.

(44) The applicant has established that this new pattern of amylase distribution is valid and applicable for detecting infection at sites.

(45) In terms of the mechanism by which amylase distribution arises at the site of infection, then there are various possibilities. Amylase which is occurring naturally in the body and distributed throughout the body could be caused to collect at the infection site.

(46) One way this could be caused to happen is through enhanced vascular permeability, and reduced lymphatic clearance: (the so-called enhanced permeability and retention effect). Histamine release has been reported to stimulate amylase secretion and increase vascular permeability, thereby augmenting the EPR effect.

(47) However, the significant, selective and specific sequestration of amylase to a focus of infection reported herein is to a greater extent than seems explained by the EPR effect.

(48) Potentially, the effect is suggested to be due to invasive infection being accompanied by constant bradykinin production which amplifies the limited enhanced vascular permeability observed in non-specific infection, such as that produced in a surgical insult.

(49) The applicant's view is that the most plausible mechanism is that once in this pharmacokinetic compartment that amylase complexes immunoglobins, resulting in entrapment. The applicant references this as Microbe-Associated Large Molecule Trapping and Aggregation phenomenon, MaLTA phenomenon.

(50) Finally, certain classes of micro-organisms secrete amylase themselves, for example C. perfringens (a cause of gas gangrene), C. tetanii (tetanus), C. difficile (hospital acquired pseudomembranous colitis) and B. cereus (food poisoning) are all within the Bacillus and Clostridium genus and those are known to be capable of prolific amylase secretion and so may cause the observed increased amylase levels around infected sites, potentially several orders of magnitude higher.

(51) The mechanism by which the amylase sequesters to the infection site is not material to the invention. The invention has demonstrated that this effect is medically indicative of infection and makes use of it, irrespective of the mechanism of sequestration, to produce clinically useful results.

(52) Initial results show that the use of amylase as a biomarker of infection at sites is a robust method and is capable of dealing with the likely variations in pH, calcium and proteolytic activity to be expected at such sites. The use of amylase as a biomarker of infection also seems capable of successful operation in the context of a wide variety of different dressings, including Negative Pressure Wound Therapy, antibiotic treatment, site and anatomical plane, together with severity of infection.

(53) The description above makes it clear that the method and the sensors used are capable of detecting infection at a site. As an extension of that, it is possible to link the concentration level of amylase observed to the level of infection; higher levels of infection generating higher concentrations of amylase at the site and in the samples.

(54) In a further extension of that method, it is possible to establish the form of the infection which is present. One possibility in this respect is that the body will generate antibodies specific to the infection caused by the specific bacteria which is the cause of the infection. These antibodies have been observed to form complexes of immunoglobulins and amylase. This means that the antibodies specific to the infection will be in the sample taken and so could be identified in parallel with the detection of the amylase concentration.

(55) Amylase has been observed to have the capacity to complex with immunoglobulins. This means that the antibodies specific to the infection will be in the sample taken and so could be identified in parallel with the detection of amylase. In one such embodiment, this could be done via multiplex sensing.

(56) The interaction between the at least a part of the sample and the sensor occurs without the need for the human or animal body from which the sample arises being present and/or without any interaction between the device and the human or animal body which is the source of the sample. The method is provided outside the normal biological context of the sample. The method is performed outside of the human or animal body and distant therefrom. The method is provided in-vitro by the device and the sensor. The invention provides an in vitro diagnostic device. The invention provides in vitro testing for the marker.

(57) Other Applications of the Detection

(58) It has been suggested that pharmaceutical compounds can be introduced to a body in an inactive form, with the inactive form arising because the pharmaceutical compound is linked in some way to another compound which inactivates it. Whilst the inactive form would be administered to the body as a whole, for instance through injection, it could be activated at the site of interest by the activity of the amylase at that site. Thus the inactive form could be designed such that amylase causes it to breakdown or amylase catalyses the breakdown such that the active compound is rendered pharmaceutically active. The analysis of the invention could be used to establish that there is sufficient amylase, by activity and/or concentration, at the site to cause the activation and/or to impact upon the dosage given or delivery method used because of the level of amylase present at the desired release location.

(59) In a similar manner, the transportation of the pharmaceutical compound as part of a combination with amylase (described further in the next section), could be inspected by the analysis of the present invention to detect for the amylase following breakdown and hence the release of the active compound. Verification of the release would be provided.

(60) Using Amylase as a Transportation Mechanism for Pharmaceutical Compounds

(61) Whilst it has been suggested that amylase which is already naturally occurring at a site could be used to turn an inactive form including a pharmaceutical compound into an active form, potentially by releasing the pharmaceutical compound, the applicant has realised that amylase itself could be used as a transportation mechanism.

(62) When a site transitions from a non-infected to an infected site, amylase moves to and accumulates at the site, as established by the applicant above. If that amylase or some portion of it was linked to a pharmaceutical compound, then the pharmaceutical compound would be preferentially transported to the infection site. The same could apply to other sites at which amylase accumulates or is drawn in other disease situations. Thus amylase offers a targeted delivery system in such cases.

(63) Once at the site, release of the pharmaceutical compound would be triggered.

(64) A variety of constructs could be used. The amylase could be linked directly to the pharmaceutical compound. The amylase could be linked to an intermediary which is in turn linked to the pharmaceutical compound.

(65) The linking could be provided using a number of bio-conjugate techniques and chemistries, including but not limited to zero-length cross-linkers homo-bifunctional or heterobifunctional spacers which can be non-biodegradable (such as polyethylglycol) or biodegradable; homogenous or heterogenous.

(66) The pharmaceutical compound could be an antibiotic, for instance for treating infection at the site where the amylase accumulates. Other pharmaceuticals could be analgesics, growth factors etc.

(67) Sensor

(68) The sensor employed in the sample testing described above can be generally categorised as a graphene label free immunosensor. In more detail it is a highly sensitive α-amylase immunosensor platform, produced via in situ electropolymerization of aniline onto a graphene support. Electropolymerization has a material role in improving the sensitivity in detection for amylase and providing lower limits of quantification. This is a significant difference when compared with the assembly of other sensors developed for other purposes, such as those disclosed in WO2015/001286. At the same time, excellent reproducibility and stability in detection are provided, as shown in FIGS. 8, 9, 10 and 11. Covalently binding an α-amylase specific antibody to a polyaniline (PANI) layer and controlling device assembly using electrochemical impedance spectroscopy (EIS) provides a highly linear response against α-amylase concentration.

(69) To improve reproducibility of absolute readings using the sensor, when such a graphene sensor is applied, the invention suggests the use of a double sensor where simultaneous or near simultaneous readings are taken, for instance with respect to the plasma versus fluid of interest. This results in a relative scale of the magnitude of the difference, and hence a solution for the problem.

(70) Further detail on the testing of the sensor for this purpose and further detailed information on the construction of the sensor is provided below.

(71) The type of sensor used in the testing of the invention is beneficial when compared with most existing devices. In most cases, such devices are far from being bedside monitoring devices for α-amylase detection. Current α-amylase assays in clinical use are laboratory based, utilize analytical equipment with a large footprint, have an appreciable turnaround time (TAT), measure activity rather than concentration, and are susceptible to hemolysis and inactivation. These substantial limitations restrict further expansion of α-amylase based diagnostics and theranostics into viable clinical practice. No use of the detection of amylase and particularly α-amylase to indicate infection has been made before.

(72) Demonstrations of Sensor Performance

(73) The performance of the sensor was explored in various ways, including: determination of amylase in a blood plasma mimic; determination of amylase in mouse plasma; and determination of amylase in human plasma.

(74) Blood Plasma Mimic

(75) The sensor was tested for its ability to measure amylase concentrations in a phosphate buffered solution that was used as a blood plasma mimic. Increasing amylase concentrations, from 1 to 500 U/L, which covers the clinically relevant range of amylase levels in the human body, were applied to individual sensors. Both Nyquist plots (FIG. 8A) and the resulting EIS calibration curve (FIG. 8B) clearly demonstrate the effective response of the immunosensor to increasing amylase concentrations.

(76) The diameter of the semicircle increased with increasing amylase concentrations demonstrating an increased resistance as a result of increased analyte concentration at the sensor surface. In general, the change in the semicircle diameter is a result of the change in the interfacial charge transfer resistance (Rct); that is, the resistance corresponding to the carrier transfer from the modified electrode to the ferricyanide in the solution. Thus, the observed diameter increase is explained as the adsorption of plasma onto anti-alpha amylase following an antigen-antibody reaction, where the adsorption of plasma effectively blocks the [Fe(CN)6].sup.3−/4− leading to an increase of Rct. The Rct in the Nyquist plot increased linearly with the amylase concentrations. This is as expected because protein structures bound to the surface of an electrode typically act as barriers to electric transfer. The average slope of the Rct versus log [amylase, U/L] was 0.348 KΩ/[amylase, U/L] with an R.sup.2 coefficient of determination of 0.93. The limit of detection (LOD) was 0.0025 U/L. This was as expected as protein structures bound to the surface of an electrode typically act as barriers to electric transfer.

(77) Mouse Plasma

(78) In order to demonstrate that the sensor function was effective when exposed to a more complex biological fluid, mouse plasma containing known quantities of amylase were used. α-amylase concentrations were measured in un-spiked and plasma samples with purified human-α-amylase. The linear response ranged from 1 to 1000 U/L and the average slope was of 0.472 KΩ[amylase, U/L]. The EIS calibration curve of the immunosensor in response to increasing concentrations of amylase in mouse plasma showed no adverse effect on sensor performance in the presence of a more complex fluid compared to the PBS (FIG. 9).

(79) Human Plasma

(80) To provide proof of function for a clinically useful device, human plasma samples obtained from a patient suffering with clinical infection were analyzed. By spiking the human plasma with known concentrations of amylase we were able to determine the effectiveness and LOD of the sensor at physiologically relevant analyte concentrations.

(81) Nyquist plots (FIG. 10) of EIS spectra show the dynamic range detectable when the sensor was exposed to various human plasma concentrations (9.9-1009.9 U/L).

(82) Overall, the sensor was demonstrated to offer highly sensitive detection and a remarkably wide limit of quantification (0.025-1000 IU/L) compared to α-amylase assays in current clinical use.

(83) In addition, the sensor had a maximum interval to obtain a result of 300 seconds. This compares very well with existing devices in clinical practice, where, despite turnaround times (TAT) being a key indicator of laboratory performance, the current TAT for clinical assays is around 45 minutes.

(84) Moreover haemolysis, sample inactivation, and the presence of contaminants interfering with colorimetric assays commonly lead to assay failure with current clinical assay systems. Since the system described in this document is label-free, and does not require either colorimetric change, it is reasonable to support the notion that the new technology will not be affected such factors.

(85) Finally, it is worth noting that the results indicate a three-log fold expansion in the lower limit of quantification when compared to current clinical assay systems. This is of interest to forensic medicine where detection of amylase can lead to a DNA profile from saliva, semen or vaginal secretions.

(86) Sensor Construction

(87) Electrochemical impedance spectroscopy (EIS) enables detection and signal output due to its ability to measure subtle changes in the electrochemical properties of materials at their interface with conducting electrodes. Gold, zinc oxide, iron oxide, and carbon are the main substrates that are being developed for use in such sensors, but carbon nanostructures, and graphene, are alternatives to gold as electrode substrates.

(88) Graphene is of particular interest as a biosensor platform due to intrinsic properties such as its large surface area, high electrical conductivity and biocompatibility. Graphene based devices have been developed that can measure minute changes in analyte concentration levels. Composed of sp.sup.2 carbon, graphene is chemically unsaturated. Intrinsically, it can undergo covalent addition to change the carbons from sp.sup.2 to spa following hybridization, however, carbon atoms in the graphene basal plane are protected by their π-conjugation system, the motion of which is constrained by surrounding carbon atoms. Therefore, basal plane covalent addition usually encounters large energy barriers, and reactive chemical groups, such as atomic hydrogen, fluorine, and pre-cursors of other chemical radicals, are usually needed as reactants. The controlled functional association of biomolecules with graphene is therefore key to developing any high throughput biosensor platform.

(89) FIG. 12 provides a schematic illustration of the construction sequence for a suitable sensor.

(90) Polyaniline (PANI) is a conductive polymer and is used as an additive transducer layer in the sensor to avoid the introduction of graphene surface defects. In addition the use of PANI improves antibody attachment to sensor electrodes while preserving optimal electrical characteristics. In addition, PANI has excellent acid/base sensitivity, a huge range of tunable conductivity, redox sensitivity, environmental stability, short reversible response times, and is easily synthesized and functionalized.

(91) The sensor of this document is an α-amylase specific immunosensor, using a combination of electro-polymerization of PANI on a graphene support and subsequent antibody binding to the polymer film.

(92) The selection of electropolymerization has a material role in improving the sensitivity in detection for amylase and providing lower limits of quantification. At the same time, excellent reproducibility and stability in detection are provided, as shown in FIGS. 8, 9, 10 and 11.

(93) The biosensor platform enables fully quantitative analysis of analyte concentrations in a simulated biological sample and in human plasma. The device displays a linear response to increasing α-amylase concentration between 1 and 1000 International Units/L (IU/L), and a LOD of 0.025 U/L. This increased sensitivity arises because of electrochemical deposition.

(94) In construction, a screen-printed graphene electrode was functionalized via polymerization with a thin film of polyaniline to provide amine groups on the graphene/PANI. The PANI film was formed by coating the electrode in a solution of aniline and subsequent electropolymerization to form a conductive polymer layer over the graphene support, enabling the transport of electron carriers to the graphene.

(95) Having deposited polyaniline, the sensor was then functionalized through covalently linking a α-amylase antibody to the PANI layer. A carbodiimide crosslinker chemistry EDAC/NHS was used for the specific activation of the —COOH terminated amino acid chains in the antibody. This forms a highly reactive O-Acylisourea intermediate that rapidly reacts with NHS to produce a stable succinimydyl ester. This ester then undergoes a nucleophilic substitution reaction with the amine groups on the PANI, leading to the formation of an orientated antibody grafted PANI layer. Due to exposure to the EDAC/NHS reagents, it is possible that each of —COOH groups at the antibody may have been activated. It is not possible therefore to ensure exclusive antibody binding through the F.sub.c region, only that orientation via the F.sub.c region is significantly higher than with random antibody adsorption. Bovine serum albumin (BSA) was then added to the sensor surface randomly, and serves to prevent any non-specific interactions with the sensor surface thereby eliminating the possibility of the sensor generating any non-specific background signal.

(96) Whilst the above is an example of a suitable sensor, other suitable sensors capable of detecting amylase activity and more preferably concentration, can be used. These include the sensors disclosed in:

(97) Federal Drug Agency (FDA) 510(k) substantial equivalence determination decision summary device only template (b) Bowling, J. L.; Katayev, A., An evaluation of the Roche Cobas c 111. Lab Medicine 2010, 41 (7), 398-402.

(98) Together with the sensors described in the following table and which are available in clinical practice. AMP=Amperometric, EIS=Electrochemical Impedance Spectroscopy, FAU=Fungal Amylase Unit. FIA=Pow Injection Analysis, KNU=kilo Novo unit, LOD=Limit of Detection, LR=Linear Range, nkat=nanokatals

(99) TABLE-US-00001 Detection Assay Design Sequential Process Technique Limits of detection Specificity Ref. Rate of Flow system using a α-glucosidase AMP LOD: 2 nkat/mL Any α- [145] product peroxide electrode and converts maltose to (0.117.64 units/mL) amylase formation enzyme membrane with α-D-glucose. when reaction time glucose oxidase, α- Mutarotase 5 min glucosidase and converts α-D- 0.5 nkat/mL optionally mutarotase glucose to β-D- (0.02941 units/mL) cross-linked by gelatin- glucose which is when reaction time glutaraldehyde determined via 30 min glucose oxidase LR: 0.1-3 mmol/L Screen-printed α-glucosidase AMP LOD: 5 units/mL Any α- [146] electrodes with converts maltose to LR: 5-250 units/mL amylase immobilized α- α-D-glucose. glucosidase, glucose Mutarotase oxidase and mutarotase converts α-D- modified with Prussian glucose to β-D- Blue glucose which is determined via glucose oxidase Flow-injection device α-glucosidase AMP LOD: Any α- [147] using maltopentaose as converts maltose to LR: 0-30 units/mL amylase substrate. α-glucosidase 5-d-glucose. immobilised on pre- Glucose oxidase activated membrane. coverts 5-d-glucose Glucose oxidase to gluconic acid and immobilized on hydrogen peroxide electrode which is measured Spectrophotometric flow Amylose incubated SIA/FIA LOD: 0.0048 FAU Any α- [148] injection measuring with sample to LR: 0.005-0.06 FAU amylase brick red complex produce maltose. formation at 540 nm 3,5 dinitrosalicylic- acid and maltose boiled Use of portable personal Sample, α- LOD: 20 U/L Any α- [149] glucose meter glucosidase and LR: 2.2-27.8 mM amylase maltopentaose incubated 15 min at 37° C. Flat-chip micro analytical Maltose Electro- LR: 0-190 kU/L Any α- [147] sensor used as part of a phosphorylase chemical amylase Micro-Electro- phosphorylates and Mechanical Systems. maltose. Glucose Lateral Pre-column and flat- oxidase converts flow enzyme electrode phosphorylated incorporated into a flow maltose to gluconic cell where maltose acid and hydrogen phosphorylase, glucose peroxide which is oxidase and peroxidase measured immobilised Colorimetric assay Disposable test AMP LR: 10-230 U/mL Any α- [150] biosensor system using strip placed under amylase Gal-G2-CNP, tongue (25 μl) chromogenic substrate Once strip inserted for α-amylase. CNP is a into reader and yellow product once saliva moved onto hydrolysed which can be the reagent paper. measured The entire test photometrically at takes roughly 30 430 nm. sec. Rate of Flow injection Sample degradation FIA LOD: 60 NU/mL Any α- [151] starch spectrophotometric of complexes LR: 0.25-5.0 amylase digestion analysis based on starch- measured in flow KNU/mL iodine complexes channel Immobilized layer of Sample placed on LR: 75-125 U/mL Any α- [152] starch gel on thick-film starch gel amylase magneto elastic sensor and presence of α- amylase alters the resonance frequency Spectrofluorimetric Sample incubated FIA LOD: 5.7 × 10.sup.−11 Any α- [153] using the quenching of with starch in flow mol/L amylase luminescence intensity channels LR: 4.8 × 10.sup.−10 − (634 nm) of nano CdS 1.2 × 10.sup.−5 mol/L doped in sol-gel of different concentrations of maltose Glycogen/amylopectin Sample incubated EIS Any α- [154] spin-coated on gold with film amylase coated quartz crystals (case frequency of 10 MHz). Films cross-linked with hexamethylene diisocyanate. Film degradation measured with quartz crystal microbalance Degradation of starch- Sample incubated LOD: 1.944 mU Any α- [155] triiodide measured using with starch- LR: 0-0.54 U amylase platinum redox sensor triiodide for direct potentiometric determination Glucose oxidase-based LOD: Any α- [156] biosensor measuring the LR: 0.66-9.83 U/mL amylase decrease in dissolved oxygen concentration related to starch concentration. Glutaraldehyde as a cross-linker Antibody to Layer of salivary The electroactive AMP LOD: 1.57 pg/mL Human [113 antigen antibody on Au- was oxidized or LR: 0.003-0.016 salivary α- electrode and reduced depending ng/mL amylase interactions monitored on concentration of by an electroactive salivary α-amylase indicator (K.sub.3Fe(CN).sub.6). present