AN ENZYME CONTAINING POLYMER, A SENSOR CONTAINING THE SAME, A MONITOR AND A MONITORING METHOD

Abstract

The present invention relates to an enzyme-containing polymer of 2-amino monosaccharide, preferably an enzyme-containing chitosan, comprising: a first repeating unit of the following Formula 1a: a second repeating unit of the following Formula 1b: and a third repeating unit of the following Formula 1c: or conjugate salts thereof, wherein all the substituents are as defined herein. There is also provided a redox polymer and the methods of preparing the enzyme-containing polymer and the redox polymer. The present invention also relates to a sensor, a method of manufacturing the sensor, a monitor, methods for monitoring failure of a tissue and uses of the sensor comprising the enzyme-containing polymer, and the monitor thereof.

##STR00001##

Claims

1. An enzyme-containing polymer comprising: a first repeating unit of Formula Ia: ##STR00039## a second repeating unit of Formula Ib: ##STR00040## and a third repeating unit of Formula Ic: ##STR00041## or conjugate salts thereof, wherein each of A, B, and D is independently a 2-amino monosaccharide; E is an enzyme comprising an n-terminal amine and optionally one or more lysine residues, wherein R.sup.2 is covalently bonded to the n-terminal amine or the amine side chain of the one or more lysine residues; Metal is a metal complex having a redox potential lower than hydrogen peroxide under physiological conditions; R.sup.1 is N*(R)(CO), N*(R)(CO)N(R), N*(R)(CR.sub.2).sub.n, N*(R)(CR.sub.2).sub.n(CO), N*(R)(CO)(CR.sub.2).sub.n, N*(R)(CO)(CR.sub.2).sub.n(CO), N*(R)(CR.sub.2).sub.nO(CR.sub.2).sub.m, N*(R)(CR.sub.2).sub.nS(CR.sub.2).sub.m, N*(R)(CR.sub.2).sub.nO, N*(R)(CR.sub.2).sub.n(CO)O, N*(R)(CO)(CR.sub.2).sub.nO, N*(R)(CO)(CR.sub.2).sub.n(CO)O, N*(R)(CR.sub.2).sub.nO(CR.sub.2).sub.mO, N*(R)(CR.sub.2).sub.nS(CR.sub.2).sub.mO, N*CH(CR.sub.2).sub.n, N*CH(CR.sub.2).sub.n(CO), N*CH(CR.sub.2).sub.nO(CR.sub.2).sub.m, N*CH(CR.sub.2).sub.nS(CR.sub.2).sub.m, N*CH(CR.sub.2).sub.nO, N*CH(CR.sub.2).sub.n(CO)O, N*CH(CR.sub.2).sub.nO(CR.sub.2).sub.mO, or N*CH(CR.sub.2).sub.nS(CR.sub.2).sub.mO, wherein N* represents the nitrogen at the 2 position of the 2-amino monosaccharide; R.sup.2 is N*(R)(CR.sub.2).sub.nN**(R), N*(R)(CR.sub.2).sub.n(CO)N**(R), N*(R)(CO)(CR.sub.2).sub.n, N*(R)(CO)(CR.sub.2).sub.n(CO)N**(R), N*(R)(CR.sub.2).sub.nO(CR.sub.2).sub.mN**(R), N*(R)(CR.sub.2).sub.nS(CR.sub.2).sub.mN**(R), N*CH(CR.sub.2).sub.nN**(H), N*CH(CR.sub.2).sub.n(CO)N**(R), N*CH(CR.sub.2).sub.nO(CR.sub.2).sub.mN**(R), N*CH(CR.sub.2).sub.nS(CR.sub.2).sub.mN**(R), N*(R)(CR.sub.2).sub.nCHN**, N*(R)(CO)(CR.sub.2).sub.nCHN**, N*(R)(CR.sub.2).sub.nO(CR.sub.2).sub.mCHN**, N*(R)(CR.sub.2).sub.nS(CR.sub.2).sub.mCHN**, N*CH(CR.sub.2).sub.nCHN**, N*CH(CR.sub.2).sub.nO(CR.sub.2).sub.mCHN**, or N*CH(CR.sub.2).sub.nS(CR.sub.2).sub.mCHN**, wherein N* represents the nitrogen at the 2 position of the 2-amino monosaccharide and N** represents the nitrogen of the n-terminal amine or the amine side chain of the one or more lysine residues of the enzyme; or R.sup.2 is represented by the moiety: ##STR00042## R.sup.3 is N*(R)(CR.sub.2).sub.nY, N*(R)(CR.sub.2).sub.n(CO)Y, N*(R)(CO)(CR.sub.2).sub.nY, N*(R)(CO)(CR.sub.2).sub.n(CO)Y, N*(R)(CR.sub.2).sub.nO(CH.sub.2).sub.mY, N*(R)(CR.sub.2).sub.nS(CR.sub.2).sub.mY, N*CH(CR.sub.2).sub.nY, N*CH(CR.sub.2).sub.n(CO)Y, N*CH(CR.sub.2).sub.nO(CR.sub.2).sub.mY, N*CH(CR.sub.2).sub.nS(CR.sub.2).sub.mY, N*(R)(CR.sub.2).sub.nCHY, N*(R)(CO)(CR.sub.2).sub.nCHY, N*(R)(CR.sub.2).sub.nO(CH.sub.2).sub.mCHY, N*(R)(CR.sub.2).sub.nS(CR.sub.2).sub.mCHY, N*CH(CR.sub.2).sub.nCHY, N*CH(CR.sub.2).sub.nO(CR.sub.2).sub.mCHY, or N*CH(CR.sub.2).sub.nS(CR.sub.2).sub.mCHY, wherein N* represents the nitrogen at the 2 position of the 2-amino monosaccharide; or R.sup.3 is represented by the moiety: ##STR00043## R for each occurrence is independently hydrogen, lower alkyl or hydroxyl; m for each occurrence is independently a whole number selected between 1-20; n for each occurrence is independently a whole number selected between 1-20; w for each occurrence is independently a whole number selected between 1-20; and Y is a polyalkylamine comprising at least one metal complex, wherein the polyalkylamine optionally crosslinks at least two of the third repeating units.

2. The enzyme-containing polymer of claim 1, wherein the metal complex is optionally substituted ferrocenyl.

3. The enzyme-containing polymer of claim 1, wherein the molar ratio of the first repeating unit to the third repeating unit in the enzyme-containing polymer is between 1:2 to 2:1.

4. The enzyme-containing polymer of claim 1, wherein the 2-amino monosaccharide is selected from the group consisting of glucosamine, mannosamine, and galactosamine.

5. The enzyme-containing polymer of claim 2, wherein the first repeating unit has the Formula IIIa: ##STR00044## the second repeating unit has the Formula IIIb: ##STR00045## and the third repeating unit has the Formula IIIc: ##STR00046## wherein R.sup.1 is (CO), (CO)(CH.sub.2).sub.n, (CO)(CH.sub.2).sub.n(CO), (CH.sub.2).sub.n(CO)O, or (CO)(CH.sub.2).sub.nO; R.sup.2 is (CH.sub.2).sub.n; R.sup.3 is (CH.sub.2).sub.n; and Y is a branched polyethylenimine comprising at least one primary or secondary amine covalently bonded to a moiety having the structure: ##STR00047##

6. The enzyme-containing polymer of claim 5, wherein R.sup.1 is (CO); R.sup.2 is (CH.sub.2).sub.3; and R.sup.3 is (CH.sub.2).sub.3.

7. The enzyme-containing polymer of claim 5 further comprising a fourth repeating unit of Formula IIId: ##STR00048##

8. The enzyme-containing polymer of claim 5, wherein the enzyme is selected from the group consisting of glucose oxidase, lactate oxidase, xanthine oxidase, cholesterol oxidase, malate oxidase, galactose oxidase, glucose dehydrogenase, lactate dehydrogenase, xanthine dehydrogenase, alcohol oxidase, choline oxidase, xanthine oxidase, glutamate oxidase and amine oxidase.

9. A redox polymer comprising a first repeating unit of Formula IIa: ##STR00049## and a second repeating unit of Formula IIb: ##STR00050## or conjugate salts thereof, wherein each of A and B independently a 2-amino monosaccharide; Metal is a metal complex having a redox potential lower than hydrogen peroxide under physiological conditions; R.sup.1 is N*(R)(CO), N*(R)(CO)N(R), N*(R)(CR.sub.2).sub.n, N*(R)(CR.sub.2).sub.n(CO), N*(R)(CO)(CR.sub.2).sub.n, N*(R)(CO)(CR.sub.2).sub.n(CO), N*(R)(CR.sub.2).sub.nO(CR.sub.2).sub.m, N*(R)(CR.sub.2).sub.nS(CR.sub.2).sub.m, N*(R)(CR.sub.2).sub.nO, N*(R)(CR.sub.2).sub.n(CO)O, N*(R)(CO)(CR.sub.2).sub.nO, N*(R)(CO)(CR.sub.2).sub.n(CO)O, N*(R)(CR.sub.2).sub.nO(CR.sub.2).sub.mO, N*(R)(CR.sub.2).sub.nS(CR.sub.2).sub.mO, N*CH(CR.sub.2).sub.n, N*CH(CR.sub.2).sub.n(CO), N*CH(CR.sub.2).sub.nO(CH.sub.2).sub.m, N*CH(CR.sub.2).sub.nS(CR.sub.2).sub.m, N*CH(CR.sub.2).sub.nO, N*CH(CR.sub.2).sub.n(CO)O, N*CH(CR.sub.2).sub.nO(CR.sub.2).sub.mO, or N*CH(CR.sub.2).sub.nS(CR.sub.2).sub.mO, wherein N* represents the nitrogen at the 2 position of the 2-amino monosaccharide; R for each occurrence is independently hydrogen or lower alkyl; m for each occurrence is independently a whole number selected between 1-20; and n for each occurrence is independently a whole number selected between 1-20.

10. The redox polymer of claim 9, wherein the first repeating unit has the Formula IVa: ##STR00051## and the second repeating unit has the Formula IVb: ##STR00052## wherein R.sup.1 is (CO), (CO)(CH.sub.2).sub.n, (CO)(CH.sub.2).sub.n(CO), (CH.sub.2).sub.n(CO)O, or (CO)(CH.sub.2).sub.nO.

11. The redox polymer of claim 10, wherein R.sup.1 is (CO).

12. A sensor, comprising: a substrate; a first sensor electrode on the substrate; a first sensing layer on the first sensor electrode, the first sensing layer comprising a first enzyme-containing polymer as defined in any one of claims 1 to 8; and a reference electrode on the substrate.

13. The sensor according to claim 12, further comprising: a second sensor electrode on the substrate; and a second sensing layer on the second sensor electrode, the second sensing layer comprising a second enzyme-containing polymer as defined in any one of claims 1 to 8.

14. The sensor according to claim 12 or 13, wherein the first sensing layer or the second sensing layer has a thickness of in the range of about 0.010 mm to about 0.300 mm.

15. The sensor according to any one of claims 12 to 14, further comprising: an elongated body portion having a body axis extending centrally through the elongated body portion; and a tip portion having a tip axis extending centrally through the tip portion, the tip portion comprising the substrate, wherein the tip portion is disposed adjacent the elongated body portion and at an obtuse angle of between 90 to 170 between the body axis and the tip axis.

16. The sensor according to claim 15, wherein the elongated body portion comprises a fluid reservoir and an actuator, and the tip portion further comprises an inflatable member, wherein a channel is disposed between the fluid reservoir and the inflatable member, and wherein the actuator is configured to deliver a volume of fluid in the fluid reservoir through the channel to the inflatable member to inflate the inflatable member.

17. The sensor according to claim 15, wherein the elongated body portion comprises a securing pin and the tip portion further comprises a moveable member, and wherein the securing pin is configured to secure the moveable member at an activated position

18. A monitor, comprising: a receiver module configured to receive a sensor output of a sensor as defined in any one of claims 12 to 17 and a control output of another sensor as defined in any one of claims 12 to 17; a processor module configured to receive a first metabolite concentration value corresponding to the sensor output and a first control value corresponding to the control output from the receiver module, wherein the processor module is configured to: compare the first metabolite concentration value against the first control value; and generate a first alarm signal on a condition that a difference between the first metabolite concentration value and the first control value is above a first pre-determined value.

19. The monitor according to claim 18, wherein the processor module is further configured to receive a second metabolite concentration value corresponding to the sensor output and a second control value corresponding to the control output from the receiver module, and wherein the processor module is further configured to: compare the second metabolite concentration value against the second control value; and generate a second alarm signal on a condition that a difference between the second metabolite concentration value and the second control value is above a second pre-determined value.

20. The monitor according to claim 19, wherein the processor module is further configured to trigger an alarm on a condition that the first alarm signal and/or the second alarm signal is generated, the alarm indicating a possibility of tissue failure.

21. The monitor according to claim 19 or 20, further comprising an output module in communication with the processor module, wherein the output module is configured to provide an indication of at least one of: the first metabolite concentration value; the first control value; the second metabolite concentration value; the second control value; the difference between the first metabolite concentration value and the first control value; the difference between the second metabolite concentration value and the second control value; the difference between the first metabolite concentration value and the first control value with respect to the first pre-determined value; and the difference between the second metabolite concentration value and the second control value with respect to the second pre-determined value.

22. A method of manufacturing a sensor, comprising: providing a substrate; forming a first sensor electrode on the substrate; forming a first sensing layer on the first sensor electrode, the first sensing layer comprising a first enzyme-containing polymer as defined in any one of claims 1 to 8; and forming a reference electrode on the substrate.

23. The method according to claim 22, further comprising: forming a second sensor electrode on the substrate; and forming a second sensing layer on the second sensor electrode, the second sensing layer comprising a second enzyme-containing polymer as defined in any one of claims 1 to 8.

24. A method for monitoring failure of a tissue on a patient, comprising the steps of: (i) providing a first sensor according to claims 12 to 17 on or within said tissue, said first sensor being capable of detecting and measuring the amount of a first metabolite; (ii) providing a second sensor according to claims 12 to 17 on a control region of said patient, said control region being separate from said tissue and wherein said second sensor is capable of detecting and measuring the amount of said first metabolite; (iii) providing a third sensor according to claims 12 to 17 on or within said tissue, said third sensor being capable of detecting and measuring the amount of a second metabolite and wherein said third sensor is the same as or different from the first sensor; (iv) providing a fourth sensor according to claims 12 to 17 on said control region of said patient, said fourth sensor being capable of detecting and measuring the amount of said second metabolite and wherein said fourth sensor is the same as or different from the second sensor; (v) monitoring the amounts of said first metabolite measured by both said first and second sensors for a period of time; and (vi) monitoring the amounts of said second metabolite measured by both said third and fourth sensors for a period of time, wherein an at least 10% decrease in the amount of said first metabolite as measured by said first sensor as compared to the amount of said first metabolite as measured by said second sensor; and at least 10% increase in the amount of said second metabolite as measured by said third sensor as compared to the amount of said second metabolite as measured by said fourth sensor, is indicative that said tissue is prone to failure.

25. A monitor, comprising: a receiver module configured to receive a sensor output of a sensor according to claims 12 to 17 and a control output of another sensor according to claims 12 to 17; a processor module configured to receive a first metabolite concentration value corresponding to the sensor output and a first control value corresponding to the control output from the receiver module, wherein the processor module is configured to: compare the first metabolite concentration value against the first control value; and generate a first alarm signal on a condition that a difference between the first metabolite concentration value and the first control value is above a first pre-determined value.

26. The monitor according to claim 25, wherein the processor module is further configured to receive a second metabolite concentration value corresponding to the sensor output and a second control value corresponding to the control output from the receiver module, and wherein the processor module is further configured to: compare the second metabolite concentration value against the second control value; and generate a second alarm signal on a condition that a difference between the second metabolite concentration value and the second control value is above a second pre-determined value.

27. The monitor according to claim 25, wherein the difference between the first metabolite concentration value and the first control value is at least 10% different from the first pre-determined value.

28. The monitor according to claim 26, wherein the difference between the second metabolite concentration value and the second control value is at least 10% different from the second pre-determined value.

29. A method for monitoring failure of a tissue on a patient, comprising the steps of: (i) providing a first sensor according to claims 12 to 17 on or within said tissue, said first sensor being capable of detecting and measuring the amount of a first metabolite; (ii) providing a second sensor according to claims 12 to 17 on a control region of said patient, said control region being separate from said tissue and wherein said second sensor is capable of detecting and measuring the amount of said first metabolite; (iii) providing a third sensor according to claims 12 to 17 on or within said tissue, said third sensor being capable of detecting and measuring the amount of a second metabolite and wherein said third sensor is the same as or different from the first sensor; (iv) providing a fourth sensor according to claims 12 to 17 on said control region of said patient, said fourth sensor being capable of detecting and measuring the amount of said second metabolite and wherein said fourth sensor is the same as or different from the second sensor; (v) monitoring the amounts of said first metabolite measured by both said first and second sensors for a period of time; and (vi) monitoring the amounts of said second metabolite measured by both said third and fourth sensors for a period of time, wherein an at least 10% decrease in the amount of said first metabolite as measured by said first sensor as compared to the amount of said first metabolite as measured by said second sensor; and at least 10% increase in the amount of said second metabolite as measured by said third sensor as compared to the amount of said second metabolite as measured by said fourth sensor, is indicative that said tissue is prone to failure.

30. A method for monitoring failure of a tissue on a patient, comprising the steps of: (i) providing a first sensor according to claims 12 to 17 on or within said tissue, said first sensor being capable of detecting and measuring the amount of a first metabolite; (ii) providing a second sensor according to claims 12 to 17 on a control region of said patient, said control region being separate from said tissue and wherein said second sensor is capable of detecting and measuring the amount of said first metabolite; (iii) providing a third sensor on according to claims 12 to 17 or within said tissue, said third sensor being capable of detecting and measuring the amount of a second metabolite and wherein said third sensor is the same as or different from the first sensor; (iv) providing a fourth sensor according to claims 12 to 17 on said control region of said patient, said fourth sensor being capable of detecting and measuring the amount of said second metabolite and wherein said fourth sensor is the same as or different from the second sensor; (v) monitoring the amounts of said first metabolite measured by both said first and second sensors for a period of time; and (vi) monitoring the amounts of said second metabolite measured by both said third and fourth sensors for a period of time, wherein the amount of said first metabolite as measured by said first sensor and the amount of said first metabolite as measured by said second sensor are substantially the same; and at least 10% increase in the amount of said second metabolite as measured by said third sensor as compared to the amount of said second metabolite as measured by said fourth sensor, is indicative that said tissue is prone to failure.

31. A method for monitoring failure of a tissue on a patient, comprising the steps of: (i) providing a first sensor according to claims 12 to 17 on or within said tissue, said first sensor being capable of detecting and measuring the amount of a first metabolite; (ii) providing a second sensor according to claims 12 to 17 on a control region of said patient, said control region being separate from said tissue and wherein said second sensor is capable of detecting and measuring the amount of said first metabolite; (iii) providing a third sensor according to claims 12 to 17 on or within said tissue, said third sensor being capable of detecting and measuring the amount of a second metabolite and wherein said third sensor is the same as or different from the first sensor; (iv) providing a fourth sensor according to claims 12 to 17 on said control region of said patient, said fourth sensor being capable of detecting and measuring the amount of said second metabolite and wherein said fourth sensor is the same as or different from the second sensor; (v) monitoring the amounts of said first metabolite measured by both said first and second sensors for a period of time; and (vi) monitoring the amounts of said second metabolite measured by both said third and fourth sensors for a period of time, wherein an at least 10% decrease in the amount of said first metabolite as measured by said first sensor as compared to the amount of said first metabolite as measured by said second sensor; and the amount of said second metabolite as measured by said third sensor and the amount of said second metabolite as measured by said fourth sensor are substantially the same, is indicative that said tissue is prone to failure.

32. A method for monitoring failure of a tissue on a patient, comprising the steps of: (i) providing a first sensor according to claims 12 to 17 on or within said tissue, said first sensor being capable of detecting and measuring the amount of a first metabolite; (ii) providing a second sensor according to claims 12 to 17 on a control region of said patient, said control region being separate from said tissue and wherein said second sensor is capable of detecting and measuring the amount of said first metabolite; (iii) providing a third sensor on according to claims 12 to 17 or within said tissue, said third sensor being capable of detecting and measuring the amount of a second metabolite and wherein said third sensor is the same as or different from the first sensor; (iv) providing a fourth sensor according to claims 12 to 17 on said control region of said patient, said fourth sensor being capable of detecting and measuring the amount of said second metabolite and wherein said fourth sensor is the same as or different from the second sensor; (v) monitoring the amounts of said first metabolite measured by both said first and second sensors for a period of time; and (vi) monitoring the amounts of said second metabolite measured by both said third and fourth sensors for a period of time, wherein an at least 10% decrease in the amount of said first metabolite as measured by said first sensor as compared to the amount of said first metabolite as measured by said second sensor; and an at least 10% decrease in the amount of said second metabolite as measured by said third sensor as compared to the amount of said second metabolite as measured by said fourth sensor, is indicative that said tissue is prone to failure.

33. A method for monitoring failure of a tissue on a patient, comprising the steps of: (i) providing a first sensor according to claims 12 to 17 on or within said tissue, said first sensor being capable of detecting and measuring the amount of a first metabolite; (ii) providing a second sensor according to claims 12 to 17 on a control region of said patient, said control region being separate from said tissue and wherein said second sensor is capable of detecting and measuring the amount of said first metabolite; (iii) providing a third sensor according to claims 12 to 17 on or within said tissue, said third sensor being capable of detecting and measuring the amount of a second metabolite and wherein said third sensor is the same as or different from the first sensor; (iv) providing a fourth sensor according to claims 12 to 17 on said control region of said patient, said fourth sensor being capable of detecting and measuring the amount of said second metabolite and wherein said fourth sensor is the same as or different from the second sensor; (v) monitoring the amounts of said first metabolite measured by both said first and second sensors for a period of time; and (vi) monitoring the amounts of said second metabolite measured by both said third and fourth sensors for a period of time, wherein an at least 10% increase in the amount of said first metabolite as measured by said first sensor as compared to the amount of said first metabolite as measured by said second sensor; and an at least 10% increase in the amount of said second metabolite as measured by said third sensor as compared to the amount of said second metabolite as measured by said fourth sensor, is indicative that said tissue is prone to failure.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0376] The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[0377] FIG. 1 shows a schematic diagram of the coupling redox reaction which occurs in a redox polymer mediated sensor.

[0378] FIG. 2 shows a schematic diagram of a sensor operating in a deflated mode, according to an example embodiment.

[0379] FIG. 3 shows a schematic diagram of a sensor operating in an inflated mode, according to an example embodiment.

[0380] FIG. 4 shows a schematic diagram (zoomed-in isometric view) of a sensor operating in a deflated mode, according to an example embodiment.

[0381] FIG. 5 shows a schematic diagram (zoomed-in isometric view) of a sensor operating in an inflated mode, according to an example embodiment.

[0382] FIG. 6 shows a schematic diagram (zoomed-in side view) of a sensor operating in a deflated mode, according to an example embodiment.

[0383] FIG. 7 shows a schematic diagram (zoomed-in side view) of a sensor operating in an inflated mode, according to an example embodiment.

[0384] FIG. 8 shows a schematic diagram (zoomed-in top view) of a sensor operating in a deflated mode, according to an example embodiment.

[0385] FIG. 9 shows a schematic diagram (zoomed-in bottom view) of a sensor operating in a deflated mode, according to an example embodiment.

[0386] FIG. 10A shows a schematic diagram (zoomed-in side view) of a sensor with a fluid reservoir operating in a deflated mode, according to an example embodiment.

[0387] FIG. 10B shows a schematic diagram (zoomed-in side view) of a sensor with a fluid reservoir operating in an inflated mode, according to an example embodiment.

[0388] FIG. 11 shows a schematic diagram of a sensor (zoomed-in exploded isometric view), according to an example embodiment.

[0389] FIG. 12A shows a schematic diagram of a tip portion (exploded isometric view), according to an example embodiment.

[0390] FIG. 12B shows a schematic diagram of a tip portion (isometric view), according to an example embodiment.

[0391] FIG. 13A shows a schematic diagram of a sensor operating in an activated mode, according to an example embodiment.

[0392] FIG. 13B shows a schematic diagram (zoomed-in isometric view) of a sensor operating in an activated mode, according to an example embodiment.

[0393] FIG. 13C shows a schematic diagram (zoomed-in side view) of a sensor operating in an activated mode, according to an example embodiment.

[0394] FIG. 13D shows a schematic diagram (zoomed-in isometric view) of a sensor operating in a deactivated mode, according to an example embodiment.

[0395] FIG. 13E shows a schematic diagram (zoomed-in side view) of a sensor operating in a deactivated mode, according to an example embodiment.

[0396] FIG. 13F shows a schematic diagram (zoomed-in isometric view) of a sensor in an intermediate mode, according to an example embodiment.

[0397] FIG. 13G shows a schematic diagram (zoomed-in side view) of a sensor in an intermediate mode, according to an example embodiment.

[0398] FIG. 14A shows a schematic diagram (zoomed-in isometric view) of a sensor, according to an example embodiment.

[0399] FIG. 14B shows a schematic diagram (zoomed-in top view) of a sensor, according to an example embodiment.

[0400] FIG. 14C shows a schematic diagram (zoomed-in side view) of a sensor, according to an example embodiment.

[0401] FIG. 14D shows a schematic diagram (zoomed-in back view) of a sensor, according to an example embodiment.

[0402] FIG. 15 shows a change in the current signal upon the dilution of 1 mM of glucose with a phosphate buffer solution.

[0403] FIG. 16 shows a change in the current signal upon the continuous addition of 100 L of 2 mM lactate into 10 mL of phosphate buffer solution (PBS) under stirring.

[0404] FIG. 17 shows the structure of chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate whereby the chitosan-ferrocenyl (CHIT-Fc) redox polymer was cross-linked with branched polyethylenimine-ferrocenyl (BPEI-Fc) intermediate via a cross-linker (1701), glutaraldehyde (GA).

[0405] FIG. 18 shows the enzyme-containing polymer whereby the enzyme may be a glucose oxidase (1801) or lactate oxidase (1803) and the cross-linker (1805).

[0406] FIG. 19 shows a voltammogram of the chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate where the current at the working electrode is plotted versus the applied voltage (that is, the working electrode's potential) to give the cyclic voltammogram trace.

[0407] FIG. 20 shows an amperometric measurement graph (current over time) to record the current change over time, for the enzyme-containing polymer (glucose oxidase), whereby the sensitivity on glucose was tested. The volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and glucose oxidase is 3:1.

[0408] FIG. 21 shows an amperometric measurement graph (current over time) to record the current change over time, for the enzyme-containing polymer (glucose oxidase), whereby the sensitivity on glucose was tested. The volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and glucose oxidase is 3:2.

[0409] FIG. 22 shows an amperometric measurement graph (current over time) to record the current change over time, for the enzyme-containing polymer (lactate oxidase), whereby the sensitivity on lactate was tested. The volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and lactate oxidase is 3:1.

[0410] FIG. 23 shows an amperometric measurement graph (current over time) to record the current change over time, for the enzyme-containing polymer (lactate oxidase), whereby the sensitivity on lactate was tested. The volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and lactate oxidase is 3:2.

[0411] FIG. 24 shows a series of photographs of the animal (rabbit) test for the glucose and lactate measurements using the sensor chip coated with the enzyme-containing polymer: FIG. 24A shows how the flap (2401) was raised and removed from the rabbit, FIG. 24B and FIG. 24C show the locations of the blood vessels supplying to the muscle and skin (2411 and 2421), FIG. 24D shows the sensor chip (2433) being wrapped around with the flap (2431), and FIG. 24E shows the blood vessel being clamped (2441) for the test.

[0412] FIG. 25 shows an amperometric measurement graph (current over time) to record the current change over time for the animal test on glucose (2501), before and after clamping (2503) of the blood vessel on the flap, at 600 seconds.

[0413] FIG. 26 shows an amperometric measurement graph (current over time) to record the current change over time for the animal test on lactate (2601), before and after clamping (2603) of the blood vessel on the flap, at 600 seconds.

DETAILED DESCRIPTION OF DRAWINGS

[0414] Referring to FIG. 2, FIG. 2 shows a schematic diagram of a sensor operating in a deflated mode, according to an example embodiment. The sensor 200 includes an elongated body portion 204 and a tip portion 202. The elongated body portion 204 includes a fluid reservoir 206. The sensor 200 further includes a connector 208. The connector 208 includes an interface for connection to a reader, a display module, or a monitor device.

[0415] Referring to FIG. 3, FIG. 3 shows a schematic diagram of a sensor operating in an inflated mode, according to an example embodiment. Similar to FIG. 2, the sensor 300 includes an elongated body portion 304 and a tip portion 302. The tip portion 302 further includes an inflatable member 312. A channel is disposed between the fluid reservoir 306 and the inflatable member 312 to provide fluid communication therebetween. The fluid reservoir 306 contains an incompressible fluid and non-toxic substance such as saline solution. An actuator 310 (shown in FIG. 3 as a clipping mechanism) is configured to deliver a volume of fluid in the fluid reservoir 306 through the channel to the inflatable member 312 to inflate the inflatable member. Comparing FIG. 2 and FIG. 3, it can be seen in FIG. 3 that the inflatable member 312 is inflated when the actuator 310 is disposed over the fluid reservoir 306. The actuator 310 provides a compressive force to move the incompressible fluid from the fluid reservoir 306 through the channel to the inflatable member 312.

[0416] Referring to FIG. 4, FIG. 4 shows a schematic diagram (zoomed-in isometric view) of a sensor operating in a deflated mode, according to an example embodiment. Similar to FIGS. 2 and 3, the sensor 400 includes an elongated body portion 404 and a tip portion 402. The tip portion 402 includes an inflatable member 412 that is deflated.

[0417] Referring to FIG. 5, FIG. 5 shows a schematic diagram (zoomed-in isometric view) of a sensor operating in an inflated mode, according to an example embodiment. Similar to FIG. 5, the sensor 500 includes an elongated body portion 504 and a tip portion 502. The tip portion 502 includes an inflatable member 512 that is inflated.

[0418] Referring to FIG. 6, FIG. 6 shows a schematic diagram (zoomed-in side view) of a sensor operating in a deflated mode, according to an example embodiment. The sensor 600 includes an elongated body portion (clearly demarcated by dashed region 604) and a tip portion (clearly demarcated by dashed region 602). The tip portion 602 includes an inflatable member 612 that is deflated. The elongated body portion 604 has a body axis 604a extending centrally through the elongated body portion 604. The tip portion 602 has a tip axis 602a extending centrally through the tip portion 602. The tip portion 602 is disposed adjacent the elongated body portion 604 and at an obtuse angle of between 90 to 170 between the body axis 604a and the tip axis 602a. More preferably, the obtuse angle between the body axis 604a and the tip axis 602a is about 130 to 160.

[0419] Referring to FIG. 7, FIG. 7 shows a schematic diagram (zoomed-in side view) of a sensor operating in an inflated mode, according to an example embodiment. The sensor 700 is similar to sensor 600, and show an inflatable member 712 that is inflated.

[0420] Referring to FIG. 8, FIG. 8 shows a schematic diagram (zoomed-in top view) of a sensor operating in a deflated mode, according to an example embodiment. Similar to FIGS. 2 to 7, the sensor 800 includes an elongated body portion (clearly demarcated by dashed region 804) and a tip portion (clearly demarcated by dashed region 802). The tip portion 802 includes an inflatable member 812 that is deflated.

[0421] Referring to FIG. 9, FIG. 9 shows a schematic diagram (zoomed-in bottom view) of a sensor operating in a deflated mode, according to an example embodiment. The tip portion 902 of the sensor comprises a substrate (not clearly seen in FIG. 9). A first sensor electrode 914, a second sensor electrode 916 and a reference electrode 918 are disposed on the substrate. The first sensor electrode 914 and the second sensor electrode 916 may comprise gold or carbon. The reference electrode 918 may comprise silver (Ag) or silver chloride (AgCl).

[0422] Referring to FIG. 10A, FIG. 10A shows a schematic diagram (zoomed-in side view) of a sensor with a fluid reservoir operating in a deflated mode, according to an example embodiment. Similar to FIG. 3, the sensor includes an elongated body portion 1004. A channel 1020 is disposed between the fluid reservoir 1006 and an inflatable member (not shown) to provide fluid communication therebetween. The fluid reservoir 1006 contains an incompressible fluid and non-toxic substance such as saline solution. The sensor further includes a connector 1008. The connector 1008 includes an interface for connection to a reader, a display module, or a monitor device (not shown).

[0423] Referring to FIG. 10B, FIG. 10B shows a schematic diagram (zoomed-in side view) of a sensor with a fluid reservoir operating in an inflated mode, according to an example embodiment. Similar to FIG. 10A, the sensor includes an elongated body portion 1004. A channel 1020 is disposed between the fluid reservoir 1006 and an inflatable member (not shown) to provide fluid communication therebetween. The fluid reservoir 1006 contains an incompressible fluid and non-toxic substance such as saline solution. The sensor further includes a connector 1008. An actuator 1010 (shown in FIG. 10B as a clipping mechanism) is configured to deliver a volume of fluid in the fluid reservoir 1006 through the channel 1020 to the inflatable member to inflate the inflatable member. When the actuator 1010 is disposed over the fluid reservoir 1006, the fluid reservoir 1006 (which is made from a resilient and flexible material) is compressed. In other words, the actuator 1010 provides a compressive force to move the incompressible fluid from the fluid reservoir 1006 through the channel 1020 to the inflatable member.

[0424] Referring to FIG. 11, FIG. 11 shows a schematic diagram of a sensor (zoomed-in exploded isometric view), according to an example embodiment. The sensor comprises a channel 1120 that is in fluid communication with inflatable member 1112. The sensor also includes a sensor body 1126 that may be made of polycarbonate. The inflatable member 1112 and a portion of the channel 1120 can be disposed within the sensor body 1126. A flexible electronics wire film 1128 provides electrical connection between electrodes at the tip portion and a reader or monitor device. An electronic wiring strain relief 1124 with integrated dielectric film may be provided.

[0425] Silver vias with silver trace 1122 may also be provided for formation of electrodes. The electrodes may be disposed within grooves formed in the sensor body 1126. The strain relief 1124 prevents the silver vias with silver trace connections 1122 from wear and tear and loss of connection when the wires 1128 are bent during insertion, securement or removal of the sensor body. The dielectric act as a medium of connection to the wires 1128 as well as insulation and protection of the silver vias with silver trace connections 1122 from external elements such as blood or other bodily fluids when the sensor is inserted, secured or removed.

[0426] Referring to FIG. 12A, FIG. 12A shows a schematic diagram of a tip portion (exploded isometric view), according to an example embodiment. A substrate 1230 provides physical support and protects the sensor electrodes, provides consistency of electrode size, polymer depth and preserves inter-electrode distance. Otherwise, sensor performance may be inconsistent and the sensors may rapidly degrade through environmental exposure. Silver electrodes and trace 1232 are provided. Silver vias 1234 are provided to enable metal contact. A polycarbonate layer 1236 may be provided. The polycarbonate layer 1236 may be about 0.5 mm thick. Furthermore, carbon electrodes 1238 and/or gold electrodes 1240 may be provided. Finally, another polycarbonate layer 1242 may be provided. The polycarbonate layer 1242 may be about 0.125 mm thick.

[0427] Referring back to FIG. 9, the carbon electrodes 1238 and/or gold electrodes 1240 correspond to the first sensor electrode 914 and/or the second sensor electrode 916. The silver electrode 1232 corresponds to the reference electrode 918.

[0428] A first sensing layer is disposed on the first sensor electrode 914. The first sensing layer comprises a first enzyme-containing polymer as described herein. A second sensing layer is disposed on the second sensor electrode 916. The second sensing layer comprises a second enzyme-containing polymer as described herein. The enzyme-containing polymer has a thickness of in the range of about 0.010 mm to about 300 mm. The enzyme-containing polymer can be deposited above carbon electrodes 1238 within the confines of the slits cut-out in the polycarbonate layer 1242.

[0429] Referring to FIG. 12B, FIG. 12B shows a schematic diagram of a tip portion (isometric view), according to an example embodiment. Similar to FIG. 12A, a substrate 1230 is provided. Silver electrodes and trace 1232 are provided. A first lower polycarbonate layer 1236 may be provided. Carbon electrodes 1238 may be provided. A second upper polycarbonate layer 1242 may be provided. The enzyme-containing polymer can be deposited above carbon electrodes 1238 within the confines of the slits cut-out in the second upper polycarbonate layer 1242.

[0430] Referring to FIG. 13A, FIG. 13A shows a schematic diagram of a sensor operating in an activated mode, according to an example embodiment. Similar to FIG. 2, the sensor 1300 includes an elongated body portion 1304 and a tip portion 1302. The elongated body portion 1304 includes an actuating arm 1306, a cable extension 1320, a biasing member 1349 and a securing pin 1350 (shown in FIG. 13C). The actuating arm 1306 is operatively coupled to the cable extension 1320, which is in turn operatively coupled to the biasing member 1349 and the securing pin 1350. The tip portion 1302 includes a moveable member 1312 and a fixed member 1348. The fixed member 1348 includes the first sensor electrode 914, the second sensor electrode 916 and the reference electrode 918 as shown in FIG. 9. Similar to FIG. 2, the sensor 1300 further includes a connector 1308. The connector 1308 includes an interface for connection to a reader, a display module, or a monitor device.

[0431] Referring to FIG. 13B, FIG. 13B shows a schematic diagram (zoomed-in isometric view) of a sensor operating in an activated mode, according to an example embodiment. The tip portion 1302 includes the fixed member 1348 and the moveable member 1312 that is in the activated mode.

[0432] Referring to FIG. 13C, FIG. 13C shows a schematic diagram (zoomed-in side view) of a sensor operating in an activated mode, according to an example embodiment. The tip portion 1302 includes the fixed member 1348 and the moveable member 1312 that is in the activated mode. The elongated body portion 1304 includes the actuating arm 1306 (not shown in FIG. 13C), the cable extension 1320, the biasing member 1349 that is in the activated mode and the securing pin 1350 that is in the activated mode.

[0433] Referring to FIG. 13D, FIG. 13D shows a schematic diagram (zoomed-in isometric view) of a sensor operating in a deactivated mode, according to an example embodiment. The tip portion 1302 includes the fixed member 1348 and the moveable member 1312 that is in the deactivated mode.

[0434] Referring to FIG. 13E, FIG. 13E shows a schematic diagram (zoomed-in side view) of a sensor operating in a deactivated mode, according to an example embodiment. The tip portion 1302 includes the fixed member 1348 and the moveable member 1312 that is in the deactivated mode. The elongated body portion 1304 includes the actuating arm 1306 (not shown in FIG. 13E), the cable extension 1320, the biasing member 1349 that is in the deactivated mode and the securing pin 1350 that is in the deactivated mode.

[0435] Referring to FIG. 13F, FIG. 13F shows a schematic diagram (zoomed-in isometric view) of a sensor in an intermediate mode, according to an example embodiment. The tip portion 1302 includes the fixed member 1348 and the moveable member 1312 that is in the intermediate mode between the activated mode and the deactivated mode.

[0436] Referring to FIG. 13G, FIG. 13G shows a schematic diagram (zoomed-in side view) of a sensor in an intermediate mode, according to an example embodiment. The tip portion 1302 includes the fixed member 1348 and the moveable member 1312 that is in the intermediate mode. The elongated body portion 1304 includes the actuating arm 1306 (not shown in FIG. 13G), the cable extension 1320, the biasing member 1349 that is in the intermediate mode and the securing pin 1350 that is in the intermediate mode.

[0437] In use, sensor 1300 in the deactivated mode is placed at a tissue such that the fixed member 1348 of the sensor 1300 is within the tissue and the moveable member 1312 is outside a patient's body. To secure the sensor 1300 to the patient, the moveable member 1312 is physically pressed downwards by a user such that the moveable member 1312 rotates about a pivot 1312A to the activated position. Referring to FIG. 13C, the biasing member 1349 that is in the form of a spring in this embodiment, provides a resilient force on the securing pin 1350 and causes the securing pin 1350 to extend fully in the activated mode, thereby securing the moveable member 1312 at the activated position by restricting the moveable member 1312 from rotating about the pivot 1312A.

[0438] To remove the sensor 1300 from the tissue, the actuating arm 1306 is pulled to retract the cable extension 1320, which in turn causes the biasing member 1349 to compress and the securing pin 1350 to slide along guide tracks to the deactivated position, such that the moveable member 1312 can rotate about the pivot 1312A to the deactivated position when the moveable member 1312 is physically pulled upwards by the user. Referring to FIG. 13E, the biasing member 1349 provides a resilient force on the securing pin 1350 and causes the securing pin 1350 to exert a force (see arrow at FIG. 13E) on the moveable member 1312 in the activated mode, thereby securing the moveable member 1312 at the activated position by restricting the moveable member 1312 from rotating about the pivot 1312A.

[0439] Referring to FIG. 14A, FIG. 14A shows a schematic diagram (zoomed-in isometric view) of a sensor, according to an example embodiment. Similar to FIG. 2, the sensor 1400 includes an elongated body portion 1404 (partially shown in FIG. 14A) and a tip portion 1402. The elongated body portion 1404 includes suturing through-holes 1403 with axes orthogonal to the body axis 604a (as shown in FIG. 6) which allows for deployment of sutures for the securement of the sensor 1400 to a tissue. The elongated body portion 1404 may include flanges 1460 with suturing through-holes 1403. Preferably, the diameter of the suturing through-holes 1403 is at least 0.3 mm Similar to FIG. 2, the sensor 1400 further includes a connector (not shown in FIG. 14A). The connector includes an interface for connection to a reader, a display module, or a monitor device.

[0440] Referring to FIG. 14B, FIG. 14B shows a schematic diagram (zoomed-in top view) of a sensor, according to an example embodiment.

[0441] Referring to FIG. 14C, FIG. 14C shows a schematic diagram (zoomed-in side view) of a sensor, according to an example embodiment. Similar to FIG. 6, the elongated body portion 1404 has a body axis extending centrally through the elongated body portion 1404. The tip portion 1402 has a tip axis extending centrally through the tip portion 1402. The tip portion 1402 is disposed adjacent the elongated body portion 1404 and at an obtuse angle of between 90 to 170 between the body axis and the tip axis. More preferably, the obtuse angle between the body axis and the tip axis is about 130 to 160.

[0442] Referring to FIG. 14D, FIG. 14D shows a schematic diagram (zoomed-in back view) of a sensor, according to an example embodiment.

[0443] Embodiments of the invention may also include one or more bio-compatible adhesive layers disposed at an underside of an elongated body portion and/or tip portion of a sensor, such that the bio-compatible adhesive layers can be in contact with a tissue for the securement of the sensor to the tissue through adhesive means.

[0444] It will be envisioned by a person skilled in the art that one or more of the securing mechanisms described above can be used in combination. For example, the inflatable member 312 of sensor 300 as shown in FIG. 3 can be used in combination with the suturing through-holes 1403 of sensor 1400 as shown in FIG. 14A and further used in combination with the bio-compatible adhesive layers.

[0445] Referring to FIG. 15, FIG. 15 shows a change in the current signal upon the dilution of 1 mM of glucose with a phosphate buffer solution. When the glucose level is reduced, the current signal reading may be different from the original current signal reading. FIG. 15 demonstrates that when the glucose level decreases, the current signal reading may drop accordingly. The glucose oxidase enzyme and the redox polymer were mixed together with the cross-linker and deposited onto the sensor surface. During the test, the sensor was inserted to the glucose solution and an electrochemical reaction occurred between the enzyme and glucose. The electrons released from the reaction were moving between the enzyme-containing polymer and the sensor surface, and were converted to a current signal by a potentiostat. When the glucose concentration decreases upon the dilution with PBS solution, the current signal value also decreased. For the in-vivo applications, when the tissue is failing, the glucose level will decrease and the current signal value monitored by the tissue sensor will also decrease, whereas the current signal value being monitored by the control sensor would be consistent.

[0446] Referring to FIG. 16, FIG. 16 shows a change in the current upon the continuous addition of 100 L of 2 mM lactate into 10 mL of phosphate buffer solution (PBS) under stirring. When the lactate level increases, the current signal reading may be different from the original current signal reading. FIG. 16 demonstrates that when the lactate level increases, the current signal reading may increase and remains at a certain level. The lactate oxidase enzyme and the redox polymer were mixed together with the cross-linker and deposited onto the sensor surface. During the test, the sensor was inserted to the lactate solution and an electrochemical reaction occurred between the enzyme and lactate. The electrons released from the reaction were moving between the enzyme-containing polymer and the sensor surface, and were converted to a current signal by a potentiostat. When the lactate concentration increases, the current signal value also increased.

[0447] For the in-vivo applications, when the tissue is failing, the lactate level will increase and the current signal value monitored by the tissue sensor will also increase, whereas the current signal value being monitored by the control sensor would be consistent.

[0448] Referring to FIG. 17, FIG. 17 shows the structure of chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate whereby the chitosan-ferrocenyl (CHIT-Fc) redox polymer was cross-linked with branched polyethylenimine-ferrocenyl (BPEI-Fc) intermediate via a cross-linker (1701), glutaraldehyde (GA). The ratio between the deacetylated monomer unit (e1) and the monomer unit crosslinked with the branched polyethylenimine-ferrocenyl intermediate (e2) may be ranging from 1:2, 1:1 and 2:1. The monomer unit having the ferrocenyl derivative (d), the deacetylated monomer unit (e1) and the monomer unit crosslinked with the branched polyethylenimine-ferrocenyl (e2) would summed up to the total deacetylated monomer units of chitosan (b).

[0449] Referring to FIG. 18, FIG. 18 shows the enzyme-containing polymer whereby the enzyme may be a glucose oxidase (1801) or lactate oxidase (1803) and the cross-linkers (1805 and 1807). The ratio between the monomer unit having the enzyme (e1) and the monomer unit crosslinked with the branched polyethylenimine-ferrocenyl intermediate (e2) may be ranging from 1:2, 1:1 and 2:1. The monomer unit having the ferrocenyl derivative (d), the monomer unit having the enzyme (e1) and the monomer unit crosslinked with the branched polyethylenimine-ferrocenyl (e2) would summed up to the total deacetylated monomer units of chitosan (b).

[0450] Referring to FIG. 20, FIG. 20 shows an amperometric measurement graph (current over time) to record the current change over time, for the enzyme-containing polymer (glucose oxidase), whereby the sensitivity on glucose was tested. The volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and glucose oxidase is 3:1, whereby the two reactants were crosslinked by the crosslinker. The concentration of glucose oxidase was 20 mg/mL in 1PBS buffer solution. The current was recorded from 0 seconds (2001), where for the first 120 seconds (2 minutes) the current signal was considered as baseline since no metabolites were in electrolyte solution to react with formulation (redox conjugation and enzyme). After the first 120 seconds (2 minutes), glucose was added into the PBS electrolyte solution, whereby more glucose was added after every 50 seconds or 100 seconds. This addition allowed the amount of glucose to increase to 0.5 mM (2003) and 1.0 mM (2005). As the amount of glucose increased to 1.5 mM (2007), 2.0 mM (2009) and 2.5 mM (2011), the current also increased with time until the amount of glucose was 3.0 mM (2013) that the graph or the current flattens to a constant value of about 3.1 A. About 5 minutes later, in order to mimic the flap failure, 5 mL of PBS solution was added (2015), whereby the concentration of glucose decreased to 1.8 mM (2017) and that was the final concentration of glucose. When the concentration of glucose gradually declined, the current decreased over time as well.

[0451] Referring to FIG. 21, FIG. 21 shows an amperometric measurement graph (current over time) to record the current change over time, for the enzyme-containing polymer (glucose oxidase), whereby the sensitivity on glucose was tested. The volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and glucose oxidase was 3:2, whereby the two reactants were crosslinked by the crosslinker. The concentration of glucose oxidase was 20 mg/mL in 1PBS buffer solution. The current was recorded from 0 seconds (2101), where for the first 120 seconds (2 minutes) the current signal was considered as baseline since no metabolites were in electrolyte solution to react with formulation (redox conjugation and enzyme). After the first 300 seconds (5 minutes), glucose was added into the PBS electrolyte solution, whereby more glucose was added after every 50 seconds or 100 seconds. This addition allowed the amount of glucose to increase to 0.2775 mM (2103) and 0.555 mM (2105). As the amount of glucose increased to 0.8325 mM (2107), the current also increased with time until PBS solution was added at 2109 to reduce/dilute the concentration of glucose. This is to mimic the flap failure, and indeed the current decreased due to the reduced concentration of glucose or the dilutions (2111).

[0452] Referring to FIG. 22, FIG. 22 shows an amperometric measurement graph (current over time) to record the current change over time, for the enzyme-containing polymer (lactate oxidase), whereby the sensitivity on lactate was tested. The volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and lactate oxidase was 3:1, whereby the two reactants were crosslinked by the crosslinker. The concentration of lactate oxidase was 20 mg/mL in 1PBS buffer solution. The current was recorded from 0 seconds (2201), where for the first 120 seconds (2 minutes) the current signal was considered as baseline since no metabolites were in electrolyte solution to react with formulation (redox conjugation and enzyme). After the first 120 seconds (2 minutes), sodium lactate was added into the PBS electrolyte solution, whereby more sodium lactate was added after every 50 seconds or 100 seconds. This addition allowed the amount of lactate to increase to 0.024 mM (2203) and 0.048 mM (2205). As the amount of lactate increased to 0.072 mM (2207) and 0.192 mM (2209), the current also increased with time until the graph or the current flattens to a constant value of about 0.8 A. About 5 minutes later, in order to mimic the flap failure, more sodium lactate was added at 2211 and the concentration of lactate increased to 0.216 mM (2213) and increased till the final amount of approximately 0.336 mM (2215). When the concentration of sodium lactate gradually increased, the current increased over time as well.

[0453] Referring to FIG. 23, FIG. 23 shows an amperometric measurement graph (current over time) to record the current change over time, for the enzyme-containing polymer (lactate oxidase), whereby the sensitivity on lactate was tested. The volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and lactate oxidase was 3:2, whereby the two reactants were crosslinked by the crosslinker. The concentration of lactate oxidase was 20 mg/mL in 1PBS buffer solution. The current was recorded from 0 seconds (2301), where for the first 120 seconds (2 minutes) the current signal was considered as baseline since no metabolites were in electrolyte to react with formulation (redox conjugation and enzyme). After the first 600 seconds (5 minutes), sodium lactate was added into the PBS electrolyte solution, whereby more sodium lactate was added after every 50 seconds or 100 seconds. This addition allowed the amount of lactate to increase to 0.0892 mM (2303) and 0.1784 mM (2305). As the amount of lactate increased to 0.2676 mM (2307), 0.5354 mM (2309), 0.58 mM (2311) and 0.6246 mM (2313), the current also increased with time. In order to mimic the flap failure, more sodium lactate was added at 2313 and about 20 minutes later, the concentration of lactate increased to a final concentration of 0.94 mM (2315). When the concentration of sodium lactate gradually increased, the current increased over time as well.

[0454] Referring to FIG. 24, FIG. 24A shows the flap (2401) with a skin paddle of 3 cm by 5 cm being raised from the rabbit. FIG. 24B and FIG. 24C show that the skin incisions were made and the flap was islanded based on the inferior epigastric blood vessel (2411 and 2421) supplying to the muscle and skin. Bilateral flaps were designed based on blood supply from the inferior epigastric vessels (2411 and 2421) on each side of the rabbit. FIG. 24D shows the sensor chip (2433) coated with the enzyme-containing polymer being wrapped around with the flap (2431). FIG. 24E shows the blood vessel being clamped (2441) for the glucose and lactate tests.

[0455] Referring to FIG. 25, FIG. 25 shows an amperometric measurement graph (current over time) to record the current change over time for glucose (2501), before and after clamping (2503) of the blood vessel on the flap, at 600 seconds. There was a significant drop in current from about 3.75 A at 600 seconds (2503) to about 2.25 A at 1100 seconds (2505).

[0456] Referring to FIG. 26, FIG. 26 shows an amperometric measurement graph (current over time) to record the current change over time for lactate (2601), before and after clamping (2603) of the blood vessel on the flap, at 600 seconds. At about 800 seconds, the current began to increase from about 0.20 A (2605) to about 0.50 A at 850 seconds (2607).

EXAMPLES

[0457] Non-limiting examples of the disclosure will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

List of Abbreviations Used

[0458] Ace: acetone

[0459] AcOH: acetic acid

[0460] BPEI: branched polyethylenimine

[0461] CHCl.sub.3: chloroform

[0462] CHIT: chitosan

[0463] DI: deionized water

[0464] EDC.HCl: N-Ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride

[0465] Ether: diethylether

[0466] Et.sub.3N: triethylamine

[0467] EtOAc: ethyl acetate

[0468] EtOH: ethanol

[0469] FcCOOH: ferrocenecarboxylic acid

[0470] FcCOCl: chlorocarbonyl ferrocene or ferrocenoyl chloride

[0471] GA: glutaraldehyde

[0472] h: hour(s)

[0473] HCl: hydrochloric acid

[0474] IPA: iso-propanol (2-propanol)

[0475] L: litre(s)

[0476] Me: methyl

[0477] MeOH: anhydrous methanol

[0478] min: minute(s)

[0479] NaOH: sodium hydroxide

[0480] NHS: N-hydroxysuccinimide

[0481] K.sub.2CO.sub.3: potassium carbonate

[0482] PBS: phosphate-buffered saline

[0483] Sec: seconds

[0484] Materials and Methods

[0485] Chitosan flakes (from shrimp shells, minimum 75% deacetylated), glucose oxidase (EC 1.1.3.4, lyophilized powder, 200 U/mg) and lactate oxidase (from Aerococcus viridans) were purchased from Sigma-Aldrich (St. Louis, Mo., United States). Branched polyethyleneimine, glucose, sodium lactate and glutaraldehyde were obtained from Sigma-Aldrich. (6-Bromohexyl) ferrocene, ferrocenecarboxylic acid, chlorocarbonyl ferrocene and other ferrocenyl derivatives were obtained from PICHEM (Shanghai, China). Other reagents including acetic acid, hydrochloric acid, potassium carbonate, sodium hydroxide, N-hydroxysuccinimide, triethylamine and 1 N-ethyl-N-(3-dimethylaminopropyl)carbodiimide hydrochloride were also obtained from Sigma-Aldrich. Solvents including, chloroform, ethyl acetate, acetone, hexane, methanol, ethanol, isopropanol, propanol and PBS buffer solution were obtained from Sigma-Aldrich. All reagents and solvents were ACS reagent grade and were used as received unless noted otherwise. The PBS (1) buffer solution was diluted from the 10PBS stock solution, and the ready-to-use buffer solutions were kept in a 4 C. fridge to avoid bacterial contamination. When required, 5 to 20 mL of PBS (1) buffer solution was taken from the main stock and transferred to sample vial for use. Stirring was applied during the glucose and lactate analysis.

[0486] Sensitivity Test on Metabolites (Glucose and Lactate)

[0487] Sensor Chip Preparation

[0488] The sensor chip was pre-cleaned by rinsing with deionized (DI) water and ethanol (EtOH), and dried with nitrogen flow. The precursor of the enzyme-containing polymer together with the respective enzyme (conjugate CHIT-Fc/BPEI-Fc:GOx and CHIT-Fc/BPEI-Fc:LOx) were prepared in condensed solution. The volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and glucose oxidase or lactate oxidase was 3:1, whereby both reactants were crosslinked by the crosslinker. The concentration of glucose oxidase or lactate oxidase was 20 mg/mL in 1PBS buffer solution. The volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and glucose oxidase or lactate oxidase was also prepared at 3:2, whereby the two reactants were crosslinked with the same crosslinker. The concentration of glucose oxidase or lactate oxidase was also 20 mg/mL in 1PBS buffer solution. The formulation solution was carefully dropped on the working electrode of DropSens chip (i.e., round disk in center) and left to dry overnight at room temperature to form a thin layer of film on the electrode.

[0489] Testing Set-Up

[0490] The sensor chip connector cable was fixed by an iron support while the other end was connected with a potentiostat. The sensor chip (electrode pads end) was then fitted in with the connector cable and the working electrode end was immersed in the electrolyte solution.

[0491] Time control and voltage control in i-t scan, amperometry 0.4 V-0.6 V of voltage was applied to activate the reaction between redox conjugate and enzyme to produce current signals. The test period was set from 15 minutes to 2 hour on-demand

[0492] Materials and Methods for Animal Studies

[0493] For the animal studies, the species selected is rabbit and the stock is from New Zealand White, where the weight is 3 kg and the gender of the rabbit is female. The rabbit is housed in the animal facility at SingHealth Experimental Medicine Centre, Singapore. All surgeries were performed under general anaesthesia. All surgeries were performed under aseptic conditions using sterile surgical instruments. Induction antibiotics and analgesics: Enrofloxacin (5 mg/kg subcutaneously) and Carprofen (5 mg/kg BW) were given. Electrical warming pads with a temperature probe were used for all surgical procedures. Bilateral rectus abdominis musculocutaneous flap with a skin paddle of size 3 cm by 5 cm was raised from the rabbit. Skin incisions were made and the flap was islanded based on the inferior epigastric blood vessels supplying to the muscle and skin. The bilateral flaps were designed based on the blood supply from the inferior epigastric vessels on each side of the rabbit. This flap that was raised is a dermal (skin) flap that comprised fats and skin tissues, as opposed to being a skeletal muscular (muscle) flap that is comprised of mainly of muscle tissue. In this case, the skin flap would show a less pronounced rise in lactate levels after the blood supply is stopped, than a muscle flap.

Example 1

[0494] Synthesis of Hexylferrocenyl-Chitosan Redox Polymer

[0495] A 1.0% chitosan (Chi) solution is prepared by dissolving 1.0 g of chitosan flakes into 100 mL of 1.0% acetic acid and stirred for 3 hours at room temperature until complete dissolution. The solution is stored in refrigerator when not in use. Briefly, 1 mg of Chi (75% deacetylated) was added into 20 mL of isopropanol 4N sodium hydroxide solution, and the mixture was heated to reflux. (6-bromohexyl) ferrocene was slowly added into the polymer solution using a pipette. The mixture was heated to reflux under nitrogen for 4 hours; the solvent was removed under reduced pressure. The residue was repetitively washed with diethyl ether (3) and methanol (2) to remove residual impurities and then dried under vacuum. The resultant hexylferrocenyl chitosan was then dialyzed for 3 days against DI water.

[0496] Preparation of Redox Polymer-Enzyme Sensors

[0497] Redox Polymer-Glucose Oxidase Enzyme

[0498] The synthesized redox polymer was dissolved into HOAc/OAc buffer solution (pH 5) until the final concentration of polymer solution was 10 mg/mL. 10 L of 10 mg/mL redox polymer solution, 5 L of 10 mg/mL glucose oxidase (GOx) and 1 L of 1% glutaraldehyde (GA) as cross-linker were mixed together and place onto the sensor surface. The mixture was then allowed to cure for 12 hours. Finally, another layer of biofilm Nafion was coated onto the sensor surface as the protective layer.

[0499] Redox Polymer-Lactate Oxidase Enzyme

[0500] The synthesized redox polymer was dissolved into HOAc/OAc buffer solution (pH 5) until the final concentration of polymer solution was 10 mg/mL. 10 L of 10 mg/mL redox polymer solution, 5 L of 10 mg/mL lactate oxidase (LOx) solution, and 1 L of 1% glutaraldehyde (GA) as cross-linker were mixed together and place onto the sensor surface. The mixture was then allowed to cure for 12 hours. Finally, another layer of biofilm Nafion was coated onto the sensor surface as the protective layer.

[0501] The redox polymer has the structure below:

##STR00035##

[0502] The enzyme-containing polymer has the structures below:

##STR00036##

Example 2

[0503] Step One: Synthesis of Chitosan-Ferrocenyl (CHIT-Fe) Redox Polymer (Crude Product)

[0504] FcCOOH was attached to CHIT with 40% w/w grafting ratio: CHIT.HCl was dissolved in DI water, and FcCOOH was dissolved in anhydrous MeOH and activated by EDC.HCl and NHS for 30 minutes. FcCOOH/EDC HCl/NHS mixture was then added into CHIT.HCl solution dropwise. The reaction was stirred for 12 hours under inert nitrogen gas. The CHIT-Fc redox polymer was obtained by neutralizing with 0.1 M NaOH solution (pH was adjusted to pH 10), then washed with DI water and MeOH several times. The precipitation obtained was dried and then dissolved in 1 wt % acetic acid solution (5 mg/ml), as shown in Scheme 1.

[0505] Step Two: Synthesis of Branched Polyethylenimine-Ferrocenyl (BPEI-Fc) Intermediate (Crude Product)

[0506] FcCOCl was attached to BPEI with 40% w/w grafting ratio: BPEI and FcCOCl were dissolved in anhydrous MeOH in 5% w/v, separately. FcCOCl was added into BPEI solution dropwise and few drops of Et.sub.3N were added into the reaction mixture. The reaction was stirred for 12 hours under inert nitrogen gas. BPEI-Fc intermediate was obtained by precipitating in CHCl.sub.3/Hexane (1:4 in v/v) mixed solvent. The precipitation obtained was washed with MeOH and re-precipitated twice. The re-precipitated precipitation was dried and then dissolved in 1PBS (35 mg/ml), as shown in Scheme 2.

##STR00037##

##STR00038##

[0507] Step Three: Synthesis of Chitosan-Ferrocenyl/Branched Polyethylenimine-Ferrocenyl (CHIT-Fc/BPEI-Fc) Conjugate and the Enzyme-Containing Polymer

[0508] 16 uL of BPEI-Fc (dissolved in 1PBS (35 mg/mL)) was fully mixed and stirred with 120 uL of CHIT-Fc (dissolved in 1 wt % acetic acid (5 mg/mL)) in the presence of 8 uL of GA solution (25 wt % in water diluted in 1PBS in 5 mg/mL) for 2 hours at room temperature to yield the CHIT-Fc/BPEI-Fc conjugate as shown in FIG. 17.

[0509] The CHIT-Fc/BPEI-Fc conjugate was then divided into two portions and mixed with the respective enzymes (glucose oxidase and lactate oxidase) to yield the enzyme-containing polymer as indicated in FIG. 18.

Example 3

[0510] Sensitivity Test on Metabolites (Glucose and Sodium Lactate)

[0511] The sensitivity test was performed once the sensor chip was coated with a thin layer of the enzyme-containing polymer (conjugate and the respective enzyme) and dipped into the PBS electrolyte solution. The thin layer of enzyme-containing polymer was prepared as mentioned above. The PBS electrolyte solution was also prepared based on the methods as mentioned above.

[0512] For the glucose testing, two different volume ratios of chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and glucose oxidase were prepared. For the first volume ratio of 3:1, the current was measured and recorded when time is 0 seconds, where the first 120 seconds (2 minutes) is considered as baseline since no metabolites were present in the PBS electrolyte solution to react with the thin film of enzyme-containing polymer. At 120 seconds, glucose was added into the PBS electrolyte solution, followed by every 50 or 100 seconds to increase the glucose concentration. More glucose was added to the electrolyte solution until the current flattens to a constant value and to mimic flap failure, the concentration of glucose was reduced by diluting with PBS electrolyte solution (2015), as indicated in FIG. 20. The current decreases as the concentration of glucose decreases to 1.8 mM (2017). When the volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and glucose oxidase was changed to 3:2, the polymer was also coated on the chip and used for testing as indicated in FIG. 21. Likewise, the current for the first 120 seconds or more is considered as baseline. After the first 300 seconds (5 minutes), when glucose was added to the electrolyte solution, the current increased as the concentration of glucose increased until 2109 of FIG. 21, where dilution occurred after more PBS electrolyte solution was added. Once the concentration of glucose decreased, the current also decreased as indicated at 2111 of FIG. 21. The detecting sensitivity on glucose was as low as 0.28 mM per change for this formulation.

[0513] For the lactate testing, two different volume ratios of chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and lactate oxidase were prepared. For the first volume ratio of 3:1, the current was measured and recorded when time is 0 seconds, where the first 120 seconds (2 minutes) is considered as baseline since no metabolites were present in the PBS electrolyte solution to react with the thin film of enzyme-containing polymer. At 120 seconds, lactate was added into the PBS electrolyte solution, followed by every 50 or 100 seconds to increase the lactate concentration. More lactate was added to the electrolyte solution until the current flattens to a constant value and to mimic flap failure, the concentration of lactate was further increased by adding more lactate to the PBS electrolyte solution at 2211 of FIG. 22. The current continued to increase as the lactate increased to 0.336 mM (2215). When the volume ratio between chitosan-ferrocenyl/branched polyethylenimine-ferrocenyl (CHIT-Fc/BPEI-Fc) conjugate and lactate oxidase was changed to 3:2, the polymer was also coated on the chip and used for testing as indicated in FIG. 23. Likewise, the current for the first 120 seconds is considered as baseline. After about 600 seconds (5 minutes), when lactate was added to the electrolyte solution, the current increased as the concentration of lactate increased until the final concentration of 0.94 mM (2315) of FIG. 23. The detecting sensitivity on sodium lactate was as low as 0.024 mM per change for this formulation.

Example 4

[0514] Animal Studies Glucose and Lactate Measurements Using the Sensor Chip Coated with the Enzyme-Containing Polymer

[0515] Using the rabbit in the animal study, the flap (2401) was prepared based on the method as mentioned above and as indicated in FIG. 24A to FIG. 24E. To measure the glucose and lactate levels on the flap, a vessel occlusion was mimicked by cutting off the blood supply (2411 and 2421) of the flap, by clamping (2441) the area surrounding the sensor chip. The sensor chip was coated with a thin layer of the enzyme-containing polymer and was wrapped around with the flap (2433).

[0516] For the glucose sensor chip test, a standard electrochemical method, amperometry was used to record the current change after the clamp was fastened on the flap. As shown in FIG. 25, in the current-time (i-t) scan for the glucose analysis (2501), the current was recorded from 0 seconds, followed by clamping of the blood vessel being performed at 600 seconds (2503). A significant drop in current was observed almost immediately after the blood vessel in the flap was being clamped. The current decreased from about 3.75 A at 600 seconds to about 2.25 A at 1100 seconds (2505). This result indicated that once the blood supply is stopped, the amount of glucose present in the specimen would be reduced.

[0517] For the lactate sensor chip test, a standard electrochemical method, amperometry was used to record the current change after the clamp was fastened on the flap. As shown in FIG. 26, in the current-time (i-t) scan for the lactate analysis (2601), the current was recorded from 0 seconds, followed by clamping of the blood vessel being performed at 600 seconds (2603). An increase in current was observed after the blood vessel in the flap was being clamped, at about 800 seconds. The current increased from about 0.18 A at 800 seconds to about 0.50 A at 850 seconds. The delay in the current response was due to the slow accumulation of lactate in the flap after the vessel was clamped. Since this flap is a skin flap, comprising mainly fats and skin tissues, the rise in lactate level is less pronounced (i.e. there is an increment of current only at 800 seconds).

INDUSTRIAL APPLICABILITY

[0518] The disclosed enzyme-containing polymer may be used to detect the presence of a metabolite in a tissue. The disclosed enzyme-containing polymer may be used in a sensor to detect the presence of a metabolite in a tissue. Where two or more different enzyme-containing polymers are used in the sensor, the amounts of two or more metabolites can be measured, and the relationship between the amounts of the metabolites may signal whether the tissue is healthy or may fail. Thus, the enzyme-containing polymer as well as the associated sensor (or device) may be used in a clinical setting to facilitate monitoring of tissue by doctors and/or nurses. The sensor or device may be used even when the tissue is buried.

[0519] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.