DETECTION OF CREATINE LEVELS USING ENZYME COMPOSITIONS

Abstract

The invention provides compositions and systems that allow the sensitive determination of the level of creatinine in a particular solution. Through the optimisation of enzymatic methods to detect creatinine the real-time determination of creatinine levels and creatinine clearance rates are also provided, allowing the real-time monitoring of kidney function. This is considered to be useful both in the monitoring of live subjects, and in the monitoring of isolated organs, such as a kidney, intended for transplantation.

Claims

1. A sensor system comprising sarcosine oxidase and/or creatininase and/or creatinase and at least a first sensor, optionally an amperometric sensor, optionally wherein the sarcosine oxidase and/or creatininase and/or creatinase are part of a composition.

2. The sensor system according to claim 1 wherein the composition comprises any two of or all of the enzymes sarcosine oxidase, creatininase and creatinase.

3. The sensor system according to any of claim 1 or 2 comprising sarcosine oxidase, creatininase and creatinase.

4. The sensor system according to claim 2 or 3 wherein at least one, optionally two, optionally all of the enzymes are not immobilised, optionally wherein all of the enzymes are in solution.

5. The sensor system according to claim 4 wherein the sarcosine oxidase, creatininase and creatinase are in solution.

6. The sensor system according to any of claims 1 to 5 further comprising a buffer, optionally wherein the composition comprises a buffer.

7. The sensor system according to claim 6 wherein the buffer is not a phosphate buffer or PBS, and/or is not a Tris buffer, and/or is not tetraborate and/or is not HEPES.

8. The sensor system according to any of claim 6 or 7 wherein the buffer is selected from the group consisting of EPPS, HEPBS, POPSO, HEPPSO and MOBS.

9. The sensor system according to any of claims 1-8 wherein the composition or the buffer is at a pH of between 7.0-9.0, optionally between 7.3-8.95, optionally 8.5.

10. The sensor system according to any of claims 1-9 wherein the composition comprises EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0 or 50 mM EPPS at pH 8.5.

11. The sensor system according to any of claims 1-10 wherein the composition further comprises urease and/or uricase and/or means to detect Cystatin C and/or means to detect albumin.

12. The sensor system according to any of claims 1-11 wherein the creatininase is from Sorachim catalogue number CNH-311; and/or the creatinase is from Sorachim catalogue number CRH-211; and/or the sarcosine oxidase is from Sorachim catalogue number SAO-351.

13. The sensor system according to any of claims 1-12 wherein the concentration of sarcosine oxidase and/or creatininase and/or creatinase in the composition is such that in the final reaction mix the concentration of creatininase is at least 300 U/ml, and/or the concentration of creatinase is at least 120 U/ml and the concentration of sarcosine oxidase is at least 10 U/ml.

14. The sensor system according to any of claims 1-13 wherein the composition is such that the final mixed solution that results from the mixing of a sample which contains creatinine and the composition of any of the preceding claims comprises creatininase, creatinase, and sarcosine oxidase at a ratio of between 10:5:1 and 49:8:1 U/ml.

15. The sensor system according to any of claims 1-13 wherein the composition is such that the final mixed solution that results from the mixing of a sample which contains creatinine and the composition of any of the preceding claims comprises creatininase, creatinase, and sarcosine oxidase in the amounts of 600 U/ml, 300 U/ml and 60 U/ml, optionally wherein the composition is at pH 8.5.

16. The sensor system according to any of claims 1-15 comprising any one of more of a microfluidic circuit, a microfluidic device, and a microdialysis probe.

17. The sensor system according to any one of claims 1-16 further comprising a continuous flow system.

18. The sensor system according to any of claims 1-17 wherein the system further comprises means to take a sample, optionally a sample from a patient or a sample from a closed-loop isolated perfused organ, optionally a kidney, optionally wherein the sample from a patient is a microdialysate, optionally from blood, urine, plasma, tissue fluid, cerebrospinal fluid.

19. The sensor system according to any of the preceding claims arranged such that the sarcosine oxidase and/or creatininase and/or creatinase or the composition according to any one of the preceding claims is added to a sample prior to contacting the sample with the sensor, optionally wherein the sensing reagent is added more than 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 seconds, 5, 5.5, 6, 6.5, 7.5, 8, 8.5, 9, 9.5 or 10 minutes prior to contact with the sensor.

20. The sensor system of any of the preceding claims wherein the system comprises means to increase the amount of oxygen in the sample, either prior to or post addition of the sensing reagent, optionally wherein the means to increase the amount of oxygen are selected from any one or more of a: a mixer, optionally that includes baffles or serpentine zones, optionally wherein the mixer is made out of a highly permeable material such as PDMS; multiple mixing stages connected by Teflon tubing; a pressurised container.

21. The sensor system of any of the preceding claims wherein the system can detect creatinine at a concentration of less than 10 uM, optionally less than 7.5 uM, optionally less than 5 uM, optionally less than 4 uM, optionally less than 3 uM, optionally less than 2 uM, optionally less than 1 uM.

22. The sensor system according to any of the preceding claims wherein the sensor system can detect a change in creatinine concentration of less than 1 uM, or less than 2 uM or less than 3 uM or less than 4 uM, or less than 5 uM or less than 7.5 uM or less than 10 uM, against a background level of creatinine of between 40 uM to 120 uM.

23. The sensor system of any of the preceding claims wherein the system comprises means for collecting data from the sensor, optionally a PowerLab/4SP, optionally wherein the system further comprises a wireless transmitting means for transmitting the data.

24. The sensor system of any of the preceding claims wherein the system further comprises means for data analysis, optionally a computer or wearable device, optionally wherein the means for data analysis comprise means for receiving wirelessly transmitted data.

25. The sensor system of any of the preceding claims further comprising at least one waste collection receptacle, optionally wherein the volume of the waste collection receptacle is less than 10 ml, for instance less than 9.5 ml, for instance less than 9 ml, for instance less than 8.5 ml, for instance less than 8 ml, for instance less than 7.5 ml, for instance less than 7 ml, for instance less than 6.5 ml, for instance less than 6 ml, for instance less than 5.5 ml, for instance less than 5 ml, for instance less than 4.5 ml, for instance less than 4 ml, for instance less than 3.5 ml, for instance less than 3 ml, for instance less than

2. 5 ml, for instance less than 2 ml, for instance less than 1.5 ml, for instance less than 1 ml, for instance less than 0.5 ml, for instance less than 0.25 ml.

26. The sensor system of any of the preceding claims wherein the system is an ambulatory system.

27. The sensor system of any of the preceding claims wherein the system comprises the means to calculate the creatinine level/creatinine clearance rate/glomerular filtration rate.

28. The sensor system according to any of the preceding claims further comprising means to deliver an agent, optionally a contrast agent or a drug or creatinine, or creatine, or sarcosine, optionally wherein the means is a drug pump, optionally wherein the drug is selected from the group consisting of immunosuppressants; chemotherapy agents such as platinum agents; antimicrobials such as the glycopeptides vancomycin and teicoplanin, and penicillin; and opioid analgesics such as morphine, diamorphine and codeine; optionally wherein the amount of agent delivered is adjusted based on the calculated creatinine level/creatinine clearance rate/glomerular filtration rate.

29. The sensor system according to any of the preceding claims wherein the system further comprises a second sensor and optionally a second means to obtain a second sample, wherein the second sample is contacted with a second sensing reagent that comprises creatinase and sarcosine oxidase prior to detection at the second sensor, optionally wherein the system is arranged such that the second sensing reagent is added the to the second sample added more than 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 seconds, 5, 5.5, 6, 6.5, 7.5, 8, 8.5, 9, 9.5 or 10 minutes prior to contact with the sensor.

30. The sensor system according to claim 29 wherein the system comprises means to subtract the data obtained from the second sensor from the data obtained from the first sensor.

31. The sensor system according to any of the preceding claims wherein the first sensor captures data continuously.

32. The sensor system according to any of the preceding claims wherein the first sensor captures data at least every 24 hours, or at least every 22 hours, for example at least every 20 hours, for example at least every 18 hours, for example at least every 16 hours, for example at least every 14 hours, for example at least every 12 hours, for example at least every 10 hours, for example at least every 8 hours, for example at least every 6 hours, for example at least every 5 hours, for example at least every 4 hours, for example at least every 3 hours, for example at least every 2 hours for example at least every 1.5 hours, for example at least every 1 hour, for example at least every 50 minutes, for example at least every 45 minutes, for example at least every 40 minutes, for example at least every 35 minutes, for example at least every 30 minutes, for example at least very 25 minutes, for example at least every 20 minutes, for example at least every 15 minutes, for example at least every 10 minutes, for example at least every 5 minutes, for example at least every 2 minutes, for example at least every 1.5 minutes, for example at least every 60 seconds, for example at least every 45 seconds, for example at least every 30 seconds, for example at least every 15 seconds, for example at least every 10 seconds, for example at least every 5 seconds, for example at least every 2 seconds, for example at least every 1 second for example at least every 0.5 seconds.

33. A composition comprising any two of or all of the enzymes sarcosine oxidase, creatininase and creatinase.

34. The composition according to claim 33 comprising all of sarcosine oxidase, creatininase and creatinase.

35. The composition of claim 33 or 34 wherein at least one, optionally two, optionally all of the enzymes are not immobilised, optionally wherein all of the enzymes are in solution.

36. The composition according to claim 35 wherein the sarcosine oxidase, creatininase and creatinase are in solution.

37. The composition of claim 33-36 wherein the composition comprises a buffer.

38. The composition of claim 37 wherein the buffer is not a phosphate buffer or PBS, and/or is not a Tris buffer, and/or is not tetraborate and/or is not HEPES.

39. The composition of any one of claim 37 or 38 wherein the buffer is selected from the group consisting of EPPS, HEPBS, POPSO, HEPPSO and MOBS.

40. The composition of any one of claims 37-39 wherein the buffer has a pKa of between 7.0-9.0, optionally between 7.3-8.95, optionally 8.5.

41. The composition according to any one of claims 33-40 wherein the composition or the buffer is at a pH of between 7.0-9.0, optionally between 7.3-8.95, optionally 8.5.

42. The composition according to any one of claims 33-41 wherein the composition comprises EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0 or 50 mM EPPS at pH 8.5.

43. The composition of any one of claims 33-42 further comprising urease and/or uricase and/or means to detect Cystatin C and/or means to detect albumin.

44. The composition of any of the preceding claims wherein the creatininase is from Sorachim catalogue number CNH-311; and/or the creatinase is from Sorachim catalogue number CRH-211; and/or the sarcosine oxidase is from Sorachim catalogue number SAO-351.

45. The composition of any of the preceding claims wherein the concentration of creatininase and/or creatinase and/or sarcosine oxidase is such that in the final reaction mix the concentration of creatininase is at least 300 U/ml, and/or the concentration of creatinase is at least 120 U/ml and the concentration of sarcosine oxidase is at least 10 U/ml.

46. The composition of any of the preceding claims wherein the composition is such that the final mixed solution that results from the mixing of a sample which contains creatinine and the composition of any of the preceding claims comprises creatininase, creatinase, and sarcosine oxidase at a ratio of between 10:5:1 and 49:8:1 U/ml.

47. The composition of any of the preceding claims wherein the composition is such that the final mixed solution that results from the mixing of a sample which contains creatinine and the composition of any of the preceding claims comprises creatininase, creatinase, and sarcosine oxidase in the amounts of 600 U/ml, 300 U/ml and 60 U/ml, optionally wherein the composition is at pH 8.5.

48. A method for the determination of the level of creatinine in a sample from a human or animal subject, wherein the method comprises the use of the composition or sensor system according to any of the preceding claims, optionally wherein the sample is a dialysate or a microdialysate.

49. A method for the determination of the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate wherein the method comprises the use of the composition or sensor system according to any of the preceding claims, optionally wherein the sample is a dialysate or a microdialysate.

50. A method for the real-time determination of the level of the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate in a sample from a human or animal subject, wherein the method comprises the use of the composition of sensor system according to any of the preceding claims, optionally wherein the sample is a dialysate or a microdialysate.

51. A method for diagnosing a subject as having acute or chronic kidney disease, the method comprising determining the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate according to any of the preceding methods, optionally further comprising treating the subject for acute or chronic kidney disease or stopping treatment with a drug that is contraindicated or dangerous in acute or chronic kidney disease, optionally wherein the drug is selected from the group consisting of immunosuppressants; chemotherapy agents such as platinum agents; antimicrobials such as the glycopeptides vancomycin and teicoplanin, and penicillin; and opioid analgesics such as morphine, diamorphine and codeine.

52. The method of any of the preceding claims wherein determination of the level of the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate is determined following administration of an amount of creatinine and/or creatine and/or sarcosine, optionally prior to and following administration of a drug.

53. The method of any of the preceding claims wherein the method further comprises administration of a dosage of a drug, wherein the dosage has been determined based on the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate determined by the sensor system.

54. A method for monitoring a kidney for transplant, said method comprising perfusing the kidney and administering an amount of creatinine and/or creatine and/or sarcosine into the system, and determining the creatinine clearance rate using the composition and/or system and/or methods of any of the preceding claims.

55. A method for monitoring kidney function in a recipient of a transplant wherein the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate is determined by use of the composition, sensor system and/or methods of any of the preceding claims.

56. A kit comprising: any two or all of creatininase, creatinase and sarcosine oxidase; and/or a composition according to any of the preceding claims; creatinine and/or creatine and/or sarcosine; and/or at least one waste receptacle; a buffer, optionally a buffer according to any of the preceding claims; a microdialysis probe; and/or at least one, optionally at least two precision pumps.

Description

FIGURE LEGENDS

[0228] FIG. 1. Comparing 30 U/ml SAO in 10 mM PBS from pH 7.0-8.0 using a 50 m electrode.

[0229] FIG. 2. Comparing 30 U/ml SAO in 100 mM PBS at pH 7.5, 50 mM EPPS at pH 8.0, and 50 mM borate at pH 9.0 with the 825 m electrode array, demonstrating a near tripling of the current at 1 mM sarcosine in EPPS.

[0230] FIG. 3. Standardised current response profiles obtained from serial dilution experiments of 30 U/ml SAO vs. 100 uM sarcosine in various buffers, confirming the unsuitability of Tris and borate buffers for this system. Times in minutes.

[0231] FIG. 4. One of the final enzyme optimisation experiments demonstrating the normalised time evolution of the signal from the enzymatic digestion of 100 M creatinine. All enzyme amounts in Units/ml. Note the small perturbation (*) caused by a leading edge of unbuffered NaCl at pH 3.0.

[0232] FIG. 5. Creatinine calibration curve for microdialysis at 2 l/min, obtained by standard addition in well-stirred T1, with a parallel sampling curve from well-stirred defibrinated horse blood. Both curved obtained by auto-fitting to the Hill Equation.

[0233] FIG. 6. Testing for stability of the microdialysis sampling system over a 12 hour period. Note the spikes from the enzyme pump refilling every 40 minutes (20 l at 0.5 l/min). The experiment terminated just beyond the 12 hour period when the enzyme reservoir was exhausted.

[0234] FIG. 7. Testing interference by ascorbic acid (Asc), uric acid (Uric), and paracetamol (Para). Values below labels are the running total concentration. The concentration of uric acid was estimated from its maximum solubility in water at 20 C.

[0235] FIG. 8. Simulating 100 ml/min creatinine clearance with different levels of creatinine in solution. The dotted lines show the exponential curves from which the rate constants and half-lives were derived.

[0236] FIG. 9. Results of dilution experiments to simulate different degrees of renal dysfunction, from CKD1-CKD4, equivalent to creatinine clearance rates of 100 ml/min-25 ml/min.

[0237] FIG. 10. The Waters RM3 cold perfusion system configured for warm blood perfusion with an external membrane oxygenator and heat exchanger (out of frame). The microdialysis system has completed initial calibrations and is waiting for the kidney to arrive.

[0238] FIG. 11. Grey: Raw signal from the microdialysis system showing the regular electrical spikes from the RM3's pump. Black: Results of applying a Savitsky-Golay smoothing filter to the data.

[0239] FIG. 12. A time-series image of the system being tested in a real blood-perfused pig kidney. Results of the warm perfusion experiments showing an initial plateau phase followed by a steady decrease in signal magnitude following oxygenation, and the two subsequent creatinine tests.

[0240] FIG. 13. Digestion of 100 uM creatinine in NaCl at pH 3.0, 50 mM EPPS at a) pH 7.5, b) pH 8.0 and c) pH 8.5 as the running buffer.

[0241] FIG. 14. Digestion of 100 uM creatinine in NaCl at pH 3.0. a) creatininase:creatinase:sarcosine oxidase150:300:60, b) creatininase:creatinase:sarcosine oxidase300:300:60 and c) creatininase:creatinase:sarcosine oxidase600:300:60.

[0242] FIG. 15. Creatine digestion in NaCl at pH 3.0, 50 mM EPPS at a) pH 7.5, b) pH 8.0 and c) pH 8.5 as running buffer.

[0243] FIG. 16. Creatine digestion in NaCl at pH 3.0. a) creatinase to SOA ratio=150:60, b) creatinase to SOA ratio=180:60 and c) creatinase to SOA ratio=300:60.

[0244] FIG. 17. a) Digestion of 100 uM sarcosine in 50 mM EPPS and b) Signal response vs time for different concentrations of creatinase vs sarcosine oxidase when digesting 100 uM creatinase.

[0245] FIG. 18. Solubility table.

[0246] FIG. 19. Summary of the experimental conditions described in the literature for the three-enzyme amperometric detection of creatinine. CA=creatininase, CI=creatinase, SO=sarcosine oxidase, normalised to U/ml in preparatory solutions, where 1 Unit catalyses the conversion of 1 pmol of substrate per minute. *U/cm.sup.2 of electrode. **U/electrode. ***mg of enzyme, unable to perform conversion.

EXAMPLES

Example 1

Design Requirements

[0247] The overall design concept was to create a portable, low-cost, largely turn-key, miniature system for continuously sampling and assaying normal creatinine concentrations in either the blood or urine of an isolated perfused kidney of a subject, for example a patient. This requires a system capable of detecting concentrations between 60 m-120 m for blood and 7-16 mM in the urine (Table 1.1, below). Note that these blood creatinine concentrations are only 1/25th- 1/150th the concentration of blood glucose, and that there are no systems presently capable of continuous real-time creatinine monitoring in a clinical setting [33].

TABLE-US-00002 TABLE 1.1 Normal Ranges in Blood and Urine [1, 4-6] Constituent Serum Urine General Volume ~80 ml/Kg ~1.5 L/24 hrs Properties Bodyweight Osmolality 280-295 mOsm/Kg 450-900 mOsm/Kg pH 7.35-7.45 4.5-8.0 [H.sup.+] 35-45 nmol/L 1-32,000 nmol/L Protein 60-80 g/L 150 mg/24 hrs Ions Na.sup.+ 135-145 mmol/L 40-220 mmol/24 hrs K.sup.+ 3.5-5.0 mmol/L 25-120 mmol/24 hrs Ca.sup.2+ 2.0-2.5 mmol/L 2.5-7.5 mmol/24 hrs Mg.sup.2+ 0.6-0.8 mmol/L 3-4.5 mmol/24 hrs Cl.sup. 95-105 mmol/L 100-250 mmol/24 hrs Nitrogenous Urea 1.5-5 mmol/L 420-720 mmol/24 hrs Wastes Creatinine 60-120 mol/L 7-16 mmol/24 hrs Ammonia/ 10-35 mol/L 20-70 mmol/24 hrs NH.sub.4.sup.+ Uric Acid 180-480 mol/L 1.4-4.4 mmol/24 hrs Cells Erythrocytes 4-5 10.sup.12/L.sup. 0-3/HPF Leukocytes 4-9 10.sup.9/L 0-2/HPF Other Glucose 4-6 mmol/L Absent Bilirubin 2-25 mol/L Absent Ketones Absent Absent Nitrites Absent Absent /HPF= per High Powered microscope Field

[0248] We have learned through experience is that it is important to consider the detection method for the reaction product at an early stage in the design process. Both of the reaction schemes for creatinine deiminase and the more complicated 3-step process of Tsuchida and Yoda produce species that are amenable to either electrochemical or spectrophotometric quantitation. Of these two, electrochemical methods are more suited to miniaturisation owing to the problems of optical path-lengths at small scales and the creation and stability of monochromatic light io sources required for colourimetric or absorption-based detection.

Example 2

Developing the Real-Time Assay System

[0249] The glucose and lactate sampling systems developed within our laboratory leverage a combination of microdialysis, microfluidics and amperometric sensing to create robust continuous-flow real-time assay systems (see for instance, WO 2016189301).

[0250] Amperometric Sensors

[0251] Our laboratory uses a potentiostat designed by a previous PhD researcher, Dr. Chu Wang [58]. This uses the OPA129 (Texas Instruments Inc., Dallas, Tex., USA) as the transimpedance amplifier, which has a maximum input bias current of 100 fA, a current noise figure of 0.1 fA/{square root over (Hz)} and a differential input impedance of 10.sup.13. In this design, the voltage set point is applied as an inverse voltage to the counter-electrode from the PowerLab data collection system rather than a direct bias at the working electrode, so as to minimise any possible noise at the inputs of the transimpedance amplifier. The servo part of the circuit uses an OPA140 (Texas Instruments Inc., Dallas, Tex., USA) which has a low voltage offset of 120 N, an offset voltage drift of 1 V/ C., a differential input impedance of 10.sup.13, an output impedance of 16 and a gain bandwidth product of 11 MHz.

[0252] Surface Protection with Electropolymerised m-Phenylenediamine (mPD)

[0253] The final step when preparing the needle microelectrodes is to protect the working electrode from contamination and to only allow molecules on the scale of H.sub.2O.sub.2 to reach the surface. This technique has evolved from multiple reports of polymer films used to entrap enzymes by the electrode surface to form biosensors that exist in the literature, including films of nafion [64], polypyrrole [65], and polyphenol [66].

[0254] The most stable and uniform of these are formed by in-situ electropolymerisation. In this way the precise site, rate and thickness of the final film can be controlled. We have found that polymerising meta-phenylenediamine (mPD) [67] produces reproducible thin films that are closely adherent to the surface of the working electrode and sufficiently dense as to prevent larger interfering redox species from reaching the electrode surface, such as ferrocene or those commonly found in biological systems (ascorbate, urate or paracetamol (N-(4-hydroxyphenyDacetamide)) whilst still permitting H.sub.2O.sub.2 at a rate sufficient to give good response times (<1 sec).

[0255] The method is straightforward. The needle microelectrode is suspended within a 100 mM solution of mPD in 10 mM phosphate buffered saline at pH 7.4, and a voltage of +0.7V (vs. AgIAgC1) is applied to the working electrode for 20 minutes until the current diminishes to an asymptotically low level. The electrode is then held at 0V for a further 2-5 minutes before being allowed to air dry, followed by rinsing in dH.sub.2O. The quality of the mPD layer is then checked with cyclic voltammetry, wherein a good result is considered to have reduced the magnitude of the signal peak by 95%, with equal oxidation and reduction profiles and no evidence of silver contamination.

Example 3

Optimising 3 Enzyme System

[0256] All experiments used the enzymes creatininase (CNH-311; EC 3.5.2.10; 259 U/mg), creatinase (CRH-221; EC 3.5.3.3; 9.18 U/mg), and sarcosine oxidase (SAO-351; EC 1.5.3.1; 13.3 U/mg), purchased from Sorachim (Sorachim SA., Lausanne, Switzerland) who supply enzymes from Toyobo (Toyobo Co., Ltd., Osaka, Japan).

[0257] This process of refinement took a number of months to complete, exploring the optimal range of enzyme mixtures, buffers and layout of the LabSmith microfluidic system to enable robust detection of creatinine at low concentration.

[0258] There were three noticeable trends after reviewing the literature regarding the selection and optimisation of the enzyme reaction. Firstly, the majority of researchers were using biosensors, with the enzymes embedded in a matrix applied directly to various forms of electrodes. Secondly, there was very little consistency in the specific amounts of enzyme used to create sensors nor the limits of detection derived therefrom. Thirdly, all research on this system over the past 33 years has used phosphate buffered saline (PBS) as the running buffer, see FIG. 19.

[0259] Of the papers presented in FIG. 19, only [73] and [74] did not use biosensors, employing instead spectrophotometric and flow-injection-analysis with a sequence of enzyme reaction beds, respectively.

Example 4

Buffer Selection

[0260] One reason for wishing to select a buffer other than PBS was the intended use of the system for sampling from either urine or blood. Table 1.1 shows that urinary pH can be as low as 4.5 (32 mol of H.sup.+) in normal adults. I chose to over-design the system for a pH of 3, to maintain sensitivity in the face of severe ischaemia. The pK.sub.a of PBS is only 7.2, meaning that a highly concentrated buffer would be required to provide sufficient capacity to neutralise 1 mmol of H.sup.+ and maintain the pH of the dialysate within 0.1 unit of pH 8.0. This would require a PBS concentration of 100 mM, as demonstrated by using the Henderson-Hasselbalch equation as per 3.1 below, whereas a buffer with a pK.sub.a of 8.0 should only require a concentration of 20 mM to resist a pH change of 0.1 unit.

[00002]$8.0=7.2+{\log }_{1.Math.0}\left(\frac{\mathrm{Acid}}{\mathrm{Base}}\right).Math..Math.1.Math.0^{0.8}=\left(\frac{\mathrm{Acid}}{\mathrm{Base}}\right)$$\mathrm{Base}.Math..Math.\left(1+6.3095\right)=100.Math..Math.\mathrm{mM}$$\mathrm{Base}=13.68.Math..Math.\mathrm{mM}$$\mathrm{Acid}=86.32.Math..Math.\mathrm{mM}$

[0261] Buffering 1 mmol of H+ would change the ratio as follows:

[00003]$\left(\frac{86.32}{13.68}\right).fwdarw.\left(\frac{8.Math.5.3.Math.2}{1.Math.4.6.Math.8}\right)$

[0262] Back-calculation with the Henderson-Hasselbalch Equation:

[00004]$\begin{array}{c}\mathrm{pH}=.Math.7.2+{\log }_{1.Math.0}\left(\frac{8.Math.5.3.Math.2}{1.Math.4.6.Math.8}\right)\\ =.Math.7.964\end{array}$

[0263] I examined a range of alternate buffers, looking for a suitable buffer with a pKa of 8.0, low temperature susceptibility, and lack of cation complexation and identified 4-(2-Hydroxyethyl)piperazine-I-propanesulfonic acid (EPPS), an uncommon piperazine-based agent which matched all of these criteria.

[0264] Benchtop tests demonstrated that 50 mM of EPPS was able to neutralise a saline solution at pH 3.0 to a final pH of 7.7 when mixed in a 1:4 volumetric ratio with the buffered enzyme solution, versus just pH 7.5 for enzymes in 100 mM PBS.

Example 5

Optimisation Experiments

[0265] Previous work in the lab has found that a combination of perfusate flow at 2 1/min and enzyme at 0.5 1/min produce good results. I decided to work backwards from sarcosine oxidase to creatininase, directly testing and optimising each step in turn for the enzyme mixture and pH, prior to performing microdialysis experiments.

[0266] FIG. 1 shows the results of an initial set of experiments with a single 50 m electrode which I ran prior to creating my 825 m electrode, comparing the signal magnitude of 30 U/ml sarcosine oxidase in 10 mM PBS versus sarcosine from 25 M to 10 mM, confirming my suspicions that basifying the pH to 8.0 would improve the signal. These results are similar for the two-step and three-step mixtures with higher sensitivity at pH 8.0 than 7.5.

[0267] A head-to-head comparison of 30 U/ml SAO in 100 mM PBS at pH 7.5, 50 mM EPPS at pH 8.0 and 50 mM borate buffer at pH 9.0 provided the results in FIG. 2, using the newer 825 m electrode array. The broadening of the standard deviation in the EPPS signal as the concentration progresses was most likely due to a fault with the substrate pump which also appeared in later experiments, leading to its replacement.

[0268] FIG. 3 shows the stepped profiles of these serial dilution experiments, demonstrating the clear results obtained in PBS and EPPS versus those from Tris and borate buffers, further confirming their unsuitability for this system.

[0269] Thereafter followed a series of experiments to examine the time profile of the response curves to various mixtures of enzymes to achieve the maximal response in the shortest time, beyond which minimal improvements could be seen. This would indicate that the enzyme ratios were no longer limiting, merely the amount of enzyme. I decided to limit the total enzyme content of the system (in weight/volume) to that of serum albumin (400 mg/ml), but this could be pushed further in later developments. I was mindful of the possibility of encrustation within the microfluidic system, as well as increased viscosity and interference with mixing and substrate diffusion at higher protein concentrations on these scales.

[0270] All experiments were carried out with a reservoir of 100 M substrate in normal saline at pH 3.0 into which the enzyme mixture was added in the intended 1:4 volumetric ratio and then pumped past a sensor at 2.5 l/minute to reproduce the total flow of the final system. The enzyme mixtures were buffered in 50 mM EPPS at pH 7.5, 8.0 and 8.5. The extensive series results will not be reproduced here, except for FIG. 4 which was one of the final experiments wherein the SAO and creatinase content had been optimised for 100 m creatine, and this experiment was now attempting to ascertain the optimal amount of creatininase for 100 m creatinine in normal saline at pH 3.0.

[0271] Note how increasing the pH from 8.0 to 8.5 was the equivalent of doubling the amount of creatininase content from 300 U/ml to 600 U/ml (blue vs. red lines), and the increased response with a mixture of 600:300:60 at pH 8.5. The final mixture chosen for the microdialysis experiments was 600:300:60 in 50 mM EPPS at pH 8.0, but this experiment raised the possibility of using an alternative buffering agent with a higher pKa around 8.5 in future, such as HEPBS (pKa of 8.3) [94].

[0272] Table 3.4 below presents a collection of T.sub.90 levels (time to reach 90% of maximum, measured from the beginning of the upstroke) obtained by this experimental method at pH 8.0, demonstrating the evolution of the mixture.

TABLE-US-00003 TABLE 3.4 SAO T.sub.90 CRH:SAO T.sub.90 CNH:CRH:SAO T.sub.90 (U) (sec) (U) (sec) (U) (sec) 15 138 150:60 145 150:300:60 195 30 73 180:60 104 300:300:60 154 60 28 300:60 77 600:300:60 135 Results of enzyme optimisation experiments at pH 8.0 in order to achieve minimum T.sub.90 levels. The reaction time of the final mixture is highlighted in bold.

[0273] From these results I decided to implement a 3 minute delay between the Y-junction feeding the enzyme into the dialysate, and the sensor, to ensure maximum sensitivity by providing adequate mixing and reaction time.

Example 6

Microdialysis Experiments

[0274] With the enzyme quantities and buffer optimised for detecting creatinine at levels of 100 M, I moved to test the system in a simulated final setting with microdialysis. Here, a clinical-grade CMA 70 microdialysis probe (M Dialysis AB, Stockholm, Sweden) designed for deep tissue sampling, with a membrane surface area of 18.8 mm.sup.2 and cut-off of 20 kDa was suspended in well-stirred T1 solution (an extracellular fluid analog) (our stock solution contains 2.3 mM calcium chloride, 147 mM sodium chloride, and 4 mM potassium chloride in dH.sub.2O) to which was added aliquots of creatinine in a standard-addition methodology. T1 was also used as the perfusate, delivered at 2 l/min by a Harvard Apparatus PHD 2000 programmable infusion pump (Harvard Bioscience Inc., Holliston, Mass., USA), with the dialysate returning into the Y-junction of my LabSmith board to mix with the buffered enzyme mixture flowing at 0.5 l/min, followed by the delay loop and sensor. From these results it was possible to build a calibration curve for the system, which fit the Hill Equation for enzyme kinetics with a Km of 2.3 mM (1.3 mM), V.sub.max of 2.9 mM (1.0 mM) and rate constant of 0.96 M/sec (+0.05 M/sec). Interestingly, the system's Km value encompasses that of sarcosine oxidase (Km of 2.8 mM), but not creatinine (4.5 mM) or creatininase (32 mM) which could indicate that this is the rate limiting step, perhaps even due to the availability of oxygen in solution (250 m).

[0275] The same setup was then used for standard addition experiments in well-stirred defibrinated horse blood (TCS Biosciences Ltd., Botolph Claydon, Buckingham, UK) to prove that it was possible to detect micromolar quantities of creatinine in a biological fluid. The results are given in FIG. 5.

[0276] The results obtained in T1 show that this microdialysis setup, the first of its kind, is a sensitive and low-noise method for measuring creatinine, with a limit of detection of 4.3 M and tested upper range of 500 M. The Km of the curve indicates that this method could be useful up to levels around 2 mM after further testing, providing a o broad useful working range. Furthermore, the microdialysis sampling methodology only had an estimated recovery of 40%, meaning that improving recovery could push the limit of detection down to 2 M.

[0277] The results in well-stirred horse blood show that the horse had a basal creatinine level of 180 M-186 M. This is just beyond the upper range of normal for a horse (100 M-160 M), but we do not know the muscle mass or gender of the horse from which this was obtained, nor their exercise status, whereby levels can rise to 200 M [95]. It is also possible that the sample was slightly haemolysed, with erythrocyte creatine feeding into the enzyme cascade (see Table 3.5 below). The broader standard deviations of these results no doubt come from a combination of convection effects and excluded diffusion paths due to red cell mass, altering flux across the dialysis membrane in a chaotic fashion.

Example 7

Stability Testing

[0278] In order to test the long-term stability of this microdialysis system, I suspended the probe in a well-stirred pot of T1 to which was added an amount of creatinine to bring the total concentration to 100 M. The normalised results in FIG. 6 show that the system remains responsive over a 12 hour period, with the sensitivity falling to a band between 50-60% of the original signal after 9 hours (equivalent to 250 pA), but remaining constant from that point onwards. The increase in noise from the 11% hour mark was the result of colleagues coming in to work in the morning. Spectral analysis showed three major noise peaksone at 50 Hz from the power supply, a second one at 13 Hz possibly from the magnetic stirrer, and a much slower 0.2 Hz sinusoid superimposed visible over the entire dataset which could reflect convection within the stirred liquid or the screw drive of the Harvard Apparatus PHD 2000 pump.

Example 8

Interference Testing

[0279] At a bias voltage of 700 mV versus AgIAgC1, the working electrode is able to oxidise other chemicals often found in blood such as paracetamol, uric acid, and ascorbate, but these should be prevented from reaching the electrode surface by the polymerised mPD layer. The three-enzyme system will also be able to generate H.sub.2O.sub.2 from sarcosine and creatine.

[0280] The levels of these common interferents are presented below in Table 3.5.

TABLE-US-00004 TABLE 3.5 Interferent Concentration Citation Ascorbate 164 m [77] 228 M [81] Uric Acid 916 M [81] 42-744 M * [96] Paracetamol 164 M [77] 264 M [81] Sarcosine 0.6-2.76 M * [97] Creatine 25 M * [98] 38.2-68.7 M * [99] Levels used in sensor testing. * Documented normal range in serum

[0281] I did not test for creatine and sarcosine interference in the final system because the endogenous levels of sarcosine are in the low micromolar range, and those of creatine should only cause problems in the event of extensive haemolysis, as the majority is intracellular. These two substances could also be accounted for by pre-treatment, background subtraction, or a parallel sampling pathway with a different enzyme mixture.

[0282] FIG. 7 presents the results of interference testing with the addition of the target substance into a well-stirred container of T1. The ascorbate and paracetamol were added in amounts exceeding those in the literature.

[0283] Note the response to the first addition of ascorbic acid but not the second, and a similar response to the addition of uric acid, prior to the pump refilling. These may have been due to a temporary reduction in recovery as the probe tip came into contact with the inside of the small glass sample pot used for the experiment. There is no apparent interference from the second addition of ascorbate, nor paracetamol, nor any interference with the response to a second aliquot of creatinine to bring the total concentration to 200 M.

[0284] Despite these good results, I also realised that there could be a different way to discount the effects of any potential interferents in the system.

Example 9

Measuring Creatinine Clearance

[0285] Problems with measuring absolute magnitudes of responses include the need to continuously account for drift in the sensitivity and offset of the sensor as the working electrode becomes poisoned by H.sub.2O.sub.2, coated with protein, or the reference degrades. There is also a need to account for any potential interferents in the system which may give factitious results, as discussed in the previous section.

[0286] I realised that it should be possible to construct a test for the creatinine clearance itself, deriving the renal function as a rate constant rather than measuring absolute concentrations and thereby avoid all potential concerns over interferents and sensor drift, so long as the creatinine remains detectable above background. If we consider the closed loop perfusion system should contain no endogenous creatinine, it should be possible to add a known quantity of creatinine to the circulating volume at regular intervals and monitor for the decay rate as it is filtered into the urine by the working kidney with first-order kinetics. At levels above failure, the clearance should reflect the GFR, as the contribution by active tubular secretion is minimal.

[0287] I therefore constructed a series of experiments to simulate different creatinine clearance rates for known quantities of creatinine in T1 during continuous microdialysis sampling. For example, a clearance rate of 100 ml/min would bring a 1 litre sample circulating at a rate of 1/minute (equivalent to the blood circulation rate of a normal adult human (5 litres of blood at 51/min)) to half of its original concentration in five minutes. This clearance can be simulated by steadily doubling the volume of a 2 ml sample containing a known quantity of creatinine over five minutes, or at 400 l/min. I chose to recreate the clearance rates of kidneys in various states of dysfunction, from CKD1 (Stage 1 Chronic Kidney Disease) to CKD4, with clearances of 100 ml/min, 75 ml/min, 50 ml/min and 25 ml/min respectively. Table 1.2 below first introduced the correspondence between the GFR and the stages of CKD. Note that the signal decay rate during stability testing as shown in FIG. 6 would be the equivalent to a clearance rate of 2 ml/min.

TABLE-US-00005 TABLE 1.2 The 5 stages of CKD. Stages 1 and 2 have preserved function but with evidence of renal disease, such as scarring or the presence of protein or blood in the urine. Stage 5 is also known as End- Stage Renal Disease (ESRD), requiring dialysis or transplantation. Stage 1 2 3 4 5 GFR >90 60-89 30-59 15-29 <15 (ml/min/1.73 m.sup.2)

[0288] FIG. 8 shows the results of this dilution testing for three different concentrations of creatinine (100 M, 200 M and 300 M) at a simulated clearance rate of 100 ml/min.

[0289] The results for the 200 M and 300 M experiments were very similar, with time constants giving half lives of 4760.86 seconds and 4711.0 seconds, respectively. The half life for the 100 M sample was much higher at 6202.8 seconds. It is worth noting that the decay curves are reminiscent of those described by the Albery equation [100], indicating the variability of the supply of substrate to the electrode in the dialysate is perhaps the root cause of these experimental errors, as I did not control for probe placement, stirring rate nor temperature.

[0290] FIG. 9 shows the follow-up experiment to simulate different levels of CKD. Each signal has been standardised to begin at 100% to emphasise the different decay rates observed.

[0291] The half lives of these curves were derived from an exponential fit of the raw data, providing values around 13 mins 40 seconds, 16 mins 30 seconds, and 27 mins for the 75 ml/min, 50 ml/min and 25 ml/min clearance rates respectively. Whilst these do not directly correspond to the experimental design, they do follow an ordered sequence with some proportionality between the values obtained. The results are notably more stable at lower dilution rates, further implicating dialysis recovery and mixing as sources of error.

Example 10

Testing the System with a Blood-Perfused Porcine Kidney

[0292] The final experiment explored the function of this system in an isolated perfused kidney setup. To this end, I partnered with Dr. Bynvant Sandhu, a clinical researcher working at Hammersmith Hospital, one of the UK's major renal transplantation centres. Her work involved warm blood perfusion of porcine kidneys using an RM3 perfusion device (Waters Medical Systems LLC, Rochester, Minn., USA). An adult pig kidney was collected from a nearby licensed abattoir and maintained in static cold storage for 4 hours. Following this, it was connected into an RM3 perfusion device which had been reconfigured with a heat exchanger and oxygenator for warm perfusion. The autologous blood collected for the reperfusion experiment was visibly haemolysed and contained large amounts of thrombus which had to be filtered out prior to use.

[0293] After calibrating the sensor system against 100 M creatinine directly infused into the Y-junction and then via the microdialysis probe in an unstirred 100 M creatinine-T1 solution, I placed the probe tip deep into the stump of the renal vein to ensure good flow. FIG. 10 shows the experimental setup in more detail. Data was then collected over the next hour of reperfusion until the probe membrane became damaged during repositioning and the experiment had to be abandoned.

[0294] Data analysis first required the use of a Savitsky-Golay smoothing filter (2nd order polynomial with a window of 513 samples) to remove the visible electrical spikes caused by the RM3's perfusion pump, as shown in FIG. 11.

[0295] These results show an initial plateau during system setup and initial perfusion, equivalent to 300 M creatinine. This high system offset is probably due to a combination of the muscle damage from the slaughtering process, and extensive haemolysis of the autologous blood, releasing creatine into the perfusate. When perfusion first began, the blood noticeably darkened as the kidney began consuming oxygen. Opening up the oxygen supply to the membrane oxygenator quickly returned the blood to a ruby red colour and caused a sudden decrease in the signal magnitude which soon returned to the high baseline. This may have in fact reflected a sudden oxidative burst from the ischaemic kidney consuming the oxygen required for the sarcosine oxidase to function normally, or a rapid change in pH which was detected by the sensor.

[0296] The kidney then appeared to be excreting detectable metabolites at a rate equivalent to that of the previous 100 ml/min creatinine clearance experiment, with a half-life of 6523.5 seconds, with the caveat that the results may not be entirely equivalent. I then spiked the arterial reservoir of the RM3 system with two separate aliquots of 100 moles of creatinine (10 mls10 mM), producing the results seen in FIG. 12. These curves had half-lives of 27 seconds and 18 seconds respectively, indicating that these results were more likely due to dilution than clearance.

[0297] Unfortunately the experiment had to be ended before the detectable metabolites in the system had been reduced to a low steady state. In the final reperfusion system as imagined, the perfusate would comprise washed erythrocytes in an isotonic crystalloid solution without any endogenous creatinine, thus allowing for pure clearance testing.

[0298] Conclusions

[0299] This part of the project has shown that a self-contained system based upon microdialysis sampling and amperometric testing of creatinine is able to achieve a limit of detection of 4.3 M and tested upper range of 500 M, matching or exceeding those reported in the literature (Table 3.3). This performance was due to a series of improvements and optimisations I made to the potentiostat, microelectrode sensor array and the triple-enzyme system of Tsuchida and Yoda [40]. The process of electropolymerising mPD onto the working electrode also provided good protection against levels of interferents far in excess of those reported by other groups performing such testing.

[0300] In addition to the development of a real-time creatinine monitoring system (with a 3 minute delay for reaction time) I have proposed and explored a novel way to monitor renal function without sensor calibration, thereby avoiding the need to compensate for any background noise or change in sensor offset, drift, or loss of sensitivity over time. I believe that this can be achieved by measuring the time constant (or half life) of the decay curve of creatinine excretion, and have demonstrated this experimentally in a closed-loop perfusion system containing a porcine kidney.

[0301] The economics of using this microfluidic system for real-time monitoring are also favourable. Despite the continuous wastage of the enzyme used in the analysis, a week of continuous monitoring would only consume 5 ml of the 600:300:60 mixture. At current market prices for the three enzymes as of September 2016, this would amount to less than 50/week.

[0302] Future work would see the enzymes re-optimised in a buffer with higher pK.sub.a such as HEPBS, the creation of a modular microdialysis sampling probe for in-line inclusion in a perfusion circuit, and an attempt to standardise the formation of a microelectrode array within a microchannel to provide a hot-pluggable system for live creatinine monitoring. The system may also benefit from using droplet microfluidics to allow the multiplexing of multiple enzyme reactions in parallel with a common sensor whilst producing better mixing and less Taylor dispersion to reduce signal magnitude, as is probably occurring in the 3-minute delay loop. With further development, this system could also be trialled for monitoring live renal function in an intensive-care setting.

[0303] Overall, the present invention brings us closer to the goal of maintaining organs in optimal condition prior to transplantation, buying time in a setting where every second counts.

REFERENCES

[0304] 33. Randviir E P, Banks C E. Analytical methods for quantifying creatinine within biological media. Sensors and Actuators B: Chemical. 2013; 183:239-252.

[0305] 35. Myers G L, Miller W G, Coresh J, Fleming J, Greenberg N, Greene T, et al. Recommendations for improving serum creatinine measurement: a report from the Laboratory Working Group of the National Kidney Disease Education Program. Clinical Chemistry. 2006; 52(1):5-18.

[0306] 37. Panteghini M. Enzymatic assays for creatinine: time for action. Scandinavian Journal of Clinical and Laboratory Investigation. 2008; 68(sup241):84-88.

[0307] 40. Tsuchida T, Yoda K. Multi-enzyme membrane electrodes for determination of creatinine and creatine in serum. Clinical Chemistry. 1983; 29(1):51-55.

[0308] 51. Xu J C, Hunter G W, Lukco D, Liu C C, Ward B J. Novel carbon dioxide microsensor based on tin oxide nanomaterial doped with copper oxide. IEEE Sensors Journal. 2009; 9(3):235-236.

[0309] 52. Suzuki H, Arakawa H, Karube I. Performance characteristics of a urea microsensor employing a micromachined carbon dioxide electrode. Electroanalysis. 2000; 12(16):1327-1333.

[0310] 53. Halliwell B, Clement M V, Long L H. Hydrogen peroxide in the human body. FEBS letters. 2000; 486(1):10-13.

[0311] 55. Ungerstedt U, Pycock C. Functional correlates of dopamine neurotransmission. Bulletin der Schweizerischen Akademie der Medizinischen Wissenschaften. 1974; 30(1-3):44-55.

[0312] 56. Whitesides G M. The origins and the future of microfluidics. Nature. 2006; 442(7101):368-373.

[0313] 58. Wang C. Development of clinical instruments for traumatic brain injury patients: design, realisation, and performance. Imperial College London; 2015.

[0314] 64. Liu H, Ying T, Sun K, Li H, Qi D. Reagentless amperometric biosensors highly sensitive to hydrogen peroxide, glucose and lactose based on N-methyl phenazine methosulfate incorporated in a Nafion film as an electron transfer mediator between horseradish peroxidase and an electrode. Analytica Chimica Acta. 1997; 344(3):187-199.

[0315] 65. Guerrieri A, De Benedetto G, Palmisano F, Zambonin P. Electrosynthesized non-conducting polymers as permselective membranes in amperometric enzyme electrodes: a glucose biosensor based on a co-crosslinked glucose oxidase/overoxidized polypyrrole bilayer. Biosensors and Bioelectronics. 1998; 13(1):103-112.

[0316] 66. Nakabayashi Y, Wakuda M, Imai H. Amperometric Glucose Sensors Fabricated by Electrochemical Polymerization of Phenols on Carbon Paste Electrodes Containing Ferrocene as an Electron Transfer Mediator. Analytical Sciences. 1998; 14(6):1069-1076.

[0317] 67. Mitchell K M. Acetylcholine and Choline Amperometric Enzyme Sensors Characterized in Vitro and in Vivo. Analytical Chemistry. 2004 February; 76(4):1098-1106. Available from: http://dx.doi.org/10.1021/ac034757v.

[0318] 72. Khue N V, Wolff C M, Seris J L, Schwing J P. Immobilized enzyme electrode for creatinine determination in serum. Analytical Chemistry. 1991; 63(6):611-614.

[0319] 73. Weber J, Van Zanten A. Interferences in current methods for measurements of creatinine. Clinical Chemistry. 1991; 37(5):695-700.

[0320] 74. Sakslund H, Hammerich O. Effects of pH, temperature and reaction products on the performance of an immobilized creatininase-creatinase-sarcosine oxidase enzyme system for creatinine determination. Analytica Chimica Acta. 1992; 268(2):331-345.

[0321] 75. Yamato H, Ohwa M, Wernet W. A polypyrrole/three-enzyme electrode for creatinine detection. Analytical Chemistry. 1995; 67(17):2776-2780.

[0322] 76. Schneider J, Grndig B, Renneberg R, Cammann K, Madaras M, Buck R, et al. Hydrogel matrix for three enzyme entrapment in creatine/creatinine amperometric biosensing. Analytica Chimica Acta. 1996; 325(3): 161-167.

[0323] 77. M{hacek over (a)}d{hacek over (a)}ra M B, Popescu I C, Ufer S, Buck R P. Microfabricated amperometric creatine and creatinine biosensors. Analytica Chimica Acta. 1996; 319(3):335-345.

[0324] 78. M{hacek over (a)}d{hacek over (a)}ra M B, Buck R P. Miniaturized biosensors employing electropolymerized permselective films and their use for creatinine assays in human serum. Analytical Chemistry. 1996; 68(21):3832-3839.

[0325] 79. Khan G, Wernet W. A highly sensitive amperometric creatinine sensor. Analytica Chimica Acta. 1997; 351(1):151-158.

[0326] 80. Kim E J, Haruyama T, Yanagida Y, Kobatake E, Aizawa M. Disposable creatinine sensor based on thick-film hydrogen peroxide electrode system. Analytica Chimica Acta. 1999; 394(2):225-231.

[0327] 81. Shin J H, Choi Y S, Lee H J, Choi S H, Ha J, Yoon I J, et al. A planar amperometric creatinine biosensor employing an insoluble oxidizing agent for removing redox-active interferences. Analytical Chemistry. 2001; 73(24):5965-5971.

[0328] 82. Tombach B, Schneider J, Matzkies F, Schaefer R M, Chemnitius G C. Amperometric creatinine biosensor for hemodialysis patients. Clinica Chimica Acta. 2001; 312(1):129-134.

[0329] 83. Erlenktter A, Fobker M, Chemnitius G C. Biosensors and flow-through system for the determination of creatinine in hemodialysate. Analytical and Bioanalytical Chemistry. 2002; 372(2):284-292.

[0330] 84. Stefan R I, Bokretsion R G, van Staden J F, Aboul-Enein H Y. Simultaneous determination of creatine and creatinine using amperometric biosensors. Talanta. 2003; 60(6):1223-1228.

[0331] 85. Hsiue G H, Lu P L, Chen J C. Multienzyme-immobilized modified polypropylene membrane for an amperometric creatinine biosensor. Journal of Applied Polymer Science. 2004; 92(5):3126-3134.

[0332] 86. Berberich J A, Yang L W, Madura J, Bahar I, Russell A J. A stable three-enzyme creatinine biosensor. 1. Impact of structure, function and environment on PEGylated and immobilized sarcosine oxidase. Acta Biomaterialia. 2005; 1(2):173-181.

[0333] 87. Berberich J A, Yang L W, Bahar I, Russell A J. A stable three enzyme creatinine biosensor. 2. Analysis of the impact of silver ions on creatine amidinohydrolase. Acta Biomaterialia. 2005; 1(2):183-191.

[0334] 88. Berberich J A, Chan A, Boden M, Russell A J. A stable three-enzyme creatinine biosensor. 3. Immobilization of creatinine amidohydrolase and sensor development. Acta Biomaterialia. 2005; 1(2): 193-199.

[0335] 89. Staden RISv, Bokretsion R G. Simultaneous Determination of Creatine and Creatinine using Monocrystalline Diamond Paste-Based Amperometric Biosensors. Analytical Letters. 2006; 39(11):2227-2233.

[0336] 90. Nieh C H, Tsujimura S, Shirai O, Kano K. Amperometric biosensor based on reductive H2O, detection using pentacyanoferrate-bound polymer for creatinine determination. Analytica Chimica Acta. 2013; 767:128-133.

[0337] 91. Serafn V, Hernndez P, Ag L, Yez-Sedeo P, Pingarrn J. Electrochemical biosensor for creatinine based on the immobilization of creatininase, creatinase and sarcosine oxidase onto a ferrocene/horseradish peroxidase/gold nanoparticles/multi-walled carbon nanotubes/Teflon composite electrode. Electrochimica Acta. 2013; 97:175-183.

[0338] 92. Yoshimoto T, Tanaka N, Kanada N, Inoue T, Nakajima Y, Haratake M, et al. Crystal structures of creatininase reveal the substrate binding site and provide an insight into the catalytic mechanism. Journal of Molecular Biology. 2004; 337(2):399-416.

[0339] 93. Hall S B, Khudaish E A, Hart A L. Electrochemical oxidation of hydrogen peroxide at platinum electrodes. Part IV: phosphate buffer dependence. Electrochimica Acta. 1999; 44(25):4573-4582.

[0340] 94. Ferreira C M, Pinto I S, Soares E V, Soares H M. (Un)suitability of the use of pH buffers in biological, biochemical and environmental studies and their interaction with metal ionsa review. RSC Advances. 2015; 5(39):30989-31003.

[0341] 95. Judson G, Frauenfelder H, Mooney G. Biochemical Changes in Thoroughbred Racehorses Following Submaximal and Maximal Exercise. In: Equine Exercise Physiology. Granta Editions Cambridge; 1983. p. 408-415.

[0342] 96. Fang J, Alderman M H. Serum uric acid and cardiovascular mortality: the NHANES I epidemiologic follow-up study, 1971-1992. JAMA. 2000; 283(18):2404-2410.

[0343] 97. Allen R H, Stabler S P, Lindenbaum J. Serum betaine, N,N-dimethylglycine and N-methylglycine levels in patients with cobalamin and folate deficiency and related inborn errors of metabolism. Metabolism. 1993 November; 42(11):1448-1460. Available from: http://dx.doi.org/10.1016/0026-0495(93)90198-W.

[0344] 98. Schedel J M, Tanaka H, Kiyonaga A, Shindo M, Schutz Y. Acute creatine ingestion in human: consequences on serum creatine and creatinine concentrations. Life Sciences. 1999; 65(23):24632470.

[0345] 99. Verhelst J, Berwaerts J, Marescau B, Abs R, Neels H, Mahler C, et al. Serum creatine, creatinine, and other guanidino compounds in patients with thyroid dysfunction. Metabolism. 1997; 46(9):1063-1067.

[0346] 100. Albery W J, Haggett B G, Svanberg L R. The development of electrochemical sensors. In: Advanced Agricultural Instrumentation. Springer; 1986. p. 349-392.

[0347] The invention also provides the following numbered embodiments:

[0348] 1. A composition comprising any two of or all of the enzymes creatininase, creatinase and sarcosine oxidase.

[0349] 2. The composition of embodiment 1 wherein at least one, optionally two, optionally all of the enzymes are not immobilised, optionally wherein all of the enzymes are in solution.

[0350] 3. The composition of embodiment 1 wherein the composition comprises a buffer.

[0351] 4. The composition of embodiment 3 wherein the buffer is not a phosphate buffer or PBS, and/or is not a Tris buffer, and/or is not tetraborate and/or is not HEPES.

[0352] 5. The composition of any one of embodiments 3 or 4 wherein the buffer is selected from the group consisting of EPPS, HEPBS, POPSO, HEPPSO and MOBS.

[0353] 6. The composition of any one of embodiments 3-5 wherein the buffer has a pKa of between 7.0-9.0, optionally between 7.3-8.95, optionally 8.5.

[0354] 7. The composition according to any one of embodiments 1-6 wherein the composition or the buffer is at a pH of between 7.0-9.0, optionally between 7.3-8.95, optionally 8.5.

[0355] 8. The composition according to any one of embodiments 1-7 wherein the composition comprises EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0-8.5, optionally 50 mM EPPS at pH 8.0 or 50 mM EPPS at pH 8.5.

[0356] 9. The composition of any one of embodiments 1-8 further comprising urease and/or uricase and/or means to detect Cystatin C and/or means to detect albumin.

[0357] 10. The composition of any of the preceding embodiments wherein the creatininase is from Sorachim catalogue number CNH-311; and/or the creatinase is from Sorachim catalogue number CRH-211; and/or the sarcosine oxidase is from Sorachim catalogue number SAO-351.

[0358] 11. The composition of any of the preceding embodiments wherein the concentration of creatininase and/or creatinase and/or sarcosine oxidase is such that in the final reaction mix the concentration of creatininase is at least 300 U/ml, and/or the concentration of creatinase is at least 120 U/ml and the concentration of sarcosine oxidase is at least 10 U/ml.

[0359] 12. The composition of any of the preceding embodiments wherein the composition is such that the final mixed solution that results from the mixing of a sample which contains creatinine and the composition of any of the preceding embodiments comprises creatininase, creatinase, and sarcosine oxidase at a ratio of between 10:5:1 and 49:8:1 U/ml.

[0360] 13. The composition of any of the preceding embodiments wherein the composition is such that the final mixed solution that results from the mixing of a sample which contains creatinine and the composition of any of the preceding embodiments comprises creatininase, creatinase, and sarcosine oxidase in the amounts of 600 U/ml, 300 U/ml and 60 U/ml, optionally wherein the composition is at pH 8.5.

[0361] 14. A sensor system comprising creatininase and/or creatinase and/or sarcosine oxidase and at least a first sensor, optionally an amperometric sensor, optionally wherein the creatininase and/or creatinase and/or sarcosine oxidase are part of a composition according to any one of the preceding embodiments.

[0362] 15. The sensor system according to embodiment 14 comprising any one of more of a microfluidic circuit, a microfluidic device, and a microdialysis probe.

[0363] 16. The sensor system according to any one of embodiments 14 and 15 further comprising a continuous flow system.

[0364] 17. The sensor system according to any of embodiments 14-16 wherein the system further comprises means to take a sample, optionally a sample from a patient or a sample from a closed-loop isolated perfused organ, optionally a kidney, [0365] optionally wherein the sample from a patient is a microdialysate, optionally from blood, urine, plasma, tissue fluid, cerebrospinal fluid.

[0366] 18. The sensor system according to any of the preceding embodiments arranged such that the creatininase and/or creatinase and/or sarcosine oxidase or the composition according to any one of the preceding embodiments is added to a sample prior to contacting the sample with the sensor, optionally wherein the sensing reagent is added more than 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 seconds, 5, 5.5, 6, 6.5, 7.5, 8, 8.5, 9, 9.5 or 10 minutes prior to contact with the sensor.

[0367] 19. The sensor system of any of the preceding embodiments wherein the system comprises means to increase the amount of oxygen in the sample, either prior to or post addition of the sensing reagent, optionally wherein the means to increase the amount of oxygen are selected from any one or more of a:

[0368] a mixer, optionally that includes baffles or serpentine zones, optionally wherein the mixer is made out of a highly permeable material such as PDMS;

[0369] multiple mixing stages connected by Teflon tubing;

[0370] a pressurised container.

[0371] 20. The sensor system of any of the preceding embodiments wherein the system can detect creatinine at a concentration of less than 10 uM, optionally less than 7.5 uM, optionally less than 5 uM, optionally less than 4 uM, optionally less than 3 uM, optionally less than 2 uM, optionally less than 1 uM.

[0372] 21. The sensor system according to any of the preceding embodiments wherein the sensor system can detect a change in creatinine concentration of less than 1 uM, or less than 2 uM or less than 3 uM or less than 4 uM, or less than 5 uM or less than 7.5 uM or less than 10 uM, against a background level of creatinine of between 40 uM to 120 uM.

[0373] 22. The sensor system of any of the preceding embodiments wherein the system comprises means for collecting data from the sensor, optionally a PowerLab/4SP, optionally wherein the system further comprises a wireless transmitting means for transmitting the data.

[0374] 23. The sensor system of any of the preceding embodiments wherein the system further comprises means for data analysis, optionally a computer or wearable device, optionally wherein the means for data analysis comprise means for receiving wirelessly transmitted data.

[0375] 24. The sensor system of any of the preceding embodiments further comprising at least one waste collection receptacle, optionally wherein the volume of the waste collection receptacle is less than 10 ml, for instance less than 9.5 ml, for instance less than 9 ml, for instance less than 8.5 ml, for instance less than 8 ml, for instance less than 7.5 ml, for instance less than 7 ml, for instance less than 6.5 ml, for instance less than 6 ml, for instance less than 5.5 ml, for instance less than 5 ml, for instance less than 4.5 ml, for instance less than 4 ml, for instance less than 3.5 ml, for instance less than 3 ml, for instance less than 2.5 ml, for instance less than 2 ml, for instance less than 1.5 ml, for instance less than 1 ml, for instance less than 0.5 ml, for instance less than 0.25 ml.

[0376] 25. The sensor system of any of the preceding embodiments wherein the system is an ambulatory system.

[0377] 26. The sensor system of any of the preceding embodiments wherein the system comprises the means to calculate the creatinine level/creatinine clearance rate/glomerular filtration rate.

[0378] 27. The sensor system according to any of the preceding embodiments further comprising means to deliver an agent, optionally a contrast agent or a drug or creatinine, or creatine, or sarcosine, optionally wherein the means is a drug pump, [0379] optionally wherein the drug is selected from the group consisting of immunosuppressants; chemotherapy agents such as platinum agents; antimicrobials such as the glycopeptides vancomycin and teicoplanin, and penicillin; and opioid analgesics such as morphine, diamorphine and codeine; [0380] optionally wherein the amount of agent delivered is adjusted based on the calculated creatinine level/creatinine clearance rate/glomerular filtration rate.

[0381] 28. The sensor system according to any of the preceding embodiments wherein the system further comprises a second sensor and optionally a second means to obtain a second sample, wherein the second sample is contacted with a second sensing reagent that comprises creatinase and sarcosine oxidase prior to detection at the second sensor, optionally wherein the system is arranged such that the second sensing reagent is added the to the second sample added more than 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250 seconds, 5, 5.5, 6, 6.5, 7.5, 8, 8.5, 9, 9.5 or 10 minutes prior to contact with the sensor.

[0382] 29. The sensor system according to embodiment 28 wherein the system comprises means to subtract the data obtained from the second sensor from the data obtained from the first sensor.

[0383] 30. The sensor system according to any of the preceding embodiments wherein the first sensor captures data continuously.

[0384] 31. The sensor system according to any of the preceding embodiments wherein the first sensor captures data at least every 24 hours, or at least every 22 hours, for example at least every 20 hours, for example at least every 18 hours, for example at least every 16 hours, for example at least every 14 hours, for example at least every 12 hours, for example at least every 10 hours, for example at least every 8 hours, for example at least every 6 hours, for example at least every 5 hours, for example at least every 4 hours, for example at least every 3 hours, for example at least every 2 hours for example at least every 1.5 hours, for example at least every 1 hour, for example at least every 50 minutes, for example at least every 45 minutes, for example at least every 40 minutes, for example at least every 35 minutes, for example at least every 30 minutes, for example at least very 25 minutes, for example at least every 20 minutes, for example at least every 15 minutes, for example at least every 10 minutes, for example at least every 5 minutes, for example at least every 2 minutes, for example at least every 1.5 minutes, for example at least every 60 seconds, for example at least every 45 seconds, for example at least every 30 seconds, for example at least every 15 seconds, for example at least every 10 seconds, for example at least every 5 seconds, for example at least every 2 seconds, for example at least every 1 second for example at least every 0.5 seconds.

[0385] 32. A method for the determination of the level of creatinine in a sample from a human or animal subject, wherein the method comprises the use of the composition or sensor system according to any of the preceding embodiments, optionally wherein the sample is a dialysate or a microdialysate.

[0386] 33. A method for the determination of the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate wherein the method comprises the use of the composition or sensor system according to any of the preceding embodiments, optionally wherein the sample is a dialysate or a microdialysate.

[0387] 34. A method for the real-time determination of the level of the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate in a sample from a human or animal subject, wherein the method comprises the use of the composition of sensor system according to any of the preceding embodiments, optionally wherein the sample is a dialysate or a microdialysate.

[0388] 35. A method for diagnosing a subject as having acute or chronic kidney disease, the method comprising determining the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate according to any of the preceding methods, optionally further comprising treating the subject for acute or chronic kidney disease or stopping treatment with a drug that is contraindicated or dangerous in acute or chronic kidney disease, optionally wherein the drug is selected from the group consisting of [0389] immunosuppressants; chemotherapy agents such as platinum agents; antimicrobials such as the glycopeptides vancomycin and teicoplanin, and penicillin; and opioid analgesics such as morphine, diamorphine and codeine.

[0390] 36. The method of any of the preceding embodiments wherein determination of the level of the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate is determined following administration of an amount of creatinine and/or creatine and/or sarcosine, optionally prior to and following administration of a drug.

[0391] 37. The method of any of the preceding embodiments wherein the method further comprises administration of a dosage of a drug, wherein the dosage has been determined based on the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate determined by the sensor system.

[0392] 38. A method for monitoring a kidney for transplant, said method comprising perfusing the kidney and administering an amount of creatinine and/or creatine and/or sarcosine into the system, and determining the creatinine clearance rate using the composition and/or system and/or methods of any of the preceding embodiments.

[0393] 39. A method for monitoring kidney function in a recipient of a transplant wherein the creatinine level and/or the creatinine clearance rate and/or the glomerular filtration rate is determined by use of the composition, sensor system and/or methods of any of the preceding embodiments.

[0394] 40. A kit comprising: [0395] any two or all of creatininase, creatinase and sarcosine oxidase; and/or [0396] a composition according to any of the preceding embodiments; [0397] creatinine and/or creatine and/or sarcosine; and/or [0398] at least one waste receptacle; [0399] a buffer, optionally a buffer according to any of the preceding embodiments; [0400] a microdialysis probe; and/or [0401] at least one, optionally at least two precision pumps.