Biosensors with porous chromatographic membranes

• US 20020027072A1
• Filed: 07/13/2001
• Published: 03/07/2002
• Est. Priority Date: 07/21/2000
• Status: Active Grant

• Current Assignee: i-SENS, Inc.
• Original Assignee: i-SENS, Inc.
• Inventors: Kim, Moon Hwan, Cui, Gang, Nam, Hakhyun, Cha, Geun Sig, Yoo, Jae Hyun, Woo, Byung Wook, Oh, Hyun Joon
• Granted Patent: US 6,726,818 B2
• Time in Patent Office: Days
• US Class Current: 204/403.1
• CPC Class Codes: C12Q 1/004 mediator-assisted
• CPC Class Codes: C12Q 1/006 for glucose
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: G01N 27/3272 Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels optical biosensors G01N33/52
• CPC Class Codes: G01N 30/90 Plate chromatography, e.g. thin layer or paper chromatography
• CPC Class Codes: G01N 33/491 by separating the blood components G01N15/05 takes precedence

Abstract

Disclosed is a porous membrane built-in biosensor comprising (a) at least one substrate; (b) an electrode layer patterned on the substrate, consisting of an electrode system and a circuit connector; (c) an insulator, formed on parts of the electrode layer, for electrically separating the electrode system from a circuit connector; and (d) a porous membrane via the insulator on the electrode system, wherein, when a whole blood sample is introduced to the biosensor, the whole blood sample is separated into its components during the chromatographic motion through the porous membrane so that only blood plasma can be contacted with the electrode system. The porous membrane built-in biosensor is provided with a sample inlet, which allows samples to be introduced in a constant quantity to the biosensors porous membranes without pretreatment.

Claims

1. A biosensor with porous membranes, comprising:

(a) at least one substrate;

(b) an electrode layer patterned on the substrate, consisting of an electrode system and a circuit connector;

(c) an insulator, for separating the electrode system and a circuit connector formed on parts of the electrode layer; and

(d) a porous membrane covered with the surface of electrode system by the insulator;

wherein, when a whole blood sample is introduced to the biosensor, the whole blood sample is separated into its components during the chromatographic motion through the porous membrane so that only blood plasma can be contacted with the electrode system.

2. The biosensor as set forth in claim 1, further containing upper substrate comprising a protective membrane for protecting the porous membrane, formed onto the porous membrane, or a sample inlet for protecting the porous membrane as well as being introducible samples to the biosensor.

3. The biosensor as set forth in claim 1, wherein the biosensor comprises:

(a) a lower substrate;

(b) an first electrode system patterned on the lower substrate;

(c) an adhesive formed over the electrode layer exception of porous membrane to define the area of the electrode, serving as an insulator;

(d) a porous membrane being intercalated between the counter electrode 12 and the working electrode 11, fixed with an adhesive;

(e) a second electrode positioned facing on the porous membrane, and a hole of upper substrate; and

(f) an upper substrate containing a sample inlet for protecting the porous membrane as well as being introducible samples to the biosensor, wherein either the electrode system or the porous membrane has oxidase and an electron transfer mediator immobilized thereto, when a whole blood sample is introduced to the biosensor, the whole blood sample is separated into its components during the chromatographic motion through the porous membrane so that only blood plasma can be contacted with the electrode system.

4. The biosensor as set forth in claim 1, wherein said biosensor is a differential flat type with a three-electrode system, comprising:

(a) a lower substrate 10;

(b) a plurality of first spaced circuit connector 9;

formed on the lower substrate 10,

(c) three-electrode system consisting of a working electrode 11, a counter electrode 12, and a reference electrode 13, all electrodes being established on the circuit connectors at a predetermined region

(d) an insulator 15 covering the surface of the lower substrate 10 except said the electrode system and plurality of spaced first circuit connectors exposed on terminal region of the lower substrate 10;

(e) a porous membrane 16, formed on the exposed area of the electrode system, having the same dimension as the exposed area of the electrode system; and

(f) an protective membrane 22 large enough to cover the intermediate membrane for protecting porous membrane 16 via an adhesive 17 which is not applied to the electrode system,

(g) plurality of differential circuit connectors 90 formed below the lower substrate 10;

(h) a differential electrode system consisting of a differential working electrode 110, a differential counter electrode 120, and a differential reference electrode 130;

(i) a differential insulator 150;

(j) a differential porous membrane 160;

(k) a differential adhesive 170 covering the resulting structure with the exception of the porous membrane; and

(l) a differential protective membrane 220;

each of the differential component, established on symmetry with respect to the lower substrate 10, which functions in the same pattern as that of its corresponding one in the opposite module.

5. The biosensor as set forth in claim 1, wherein said biosensor is a differential flat type with a two-electrode system, comprising:

(a) a lower substrate 10;

(b) a plurality of first spaced circuit connector 9;

formed on the lower substrate 10,

(c) a two-electrode system consisting of a working electrode 11 and a counter electrode 12, all electrodes being established on the circuit connectors at a predetermined region;

(d) an insulator 15 covering the surface of the lower substrate 10 except said the electrode system and plurality of spaced first circuit connectors exposed on terminal region of the lower substrate 10;

(e) a porous membrane 16, formed on the exposed area of the electrode system, having the same dimension as the exposed area of the electrode system; and

(f) an protective membrane 22 large enough to cover the intermediate membrane for protecting porous membrane 16 via an adhesive 17 which is not applied to the electrode system,

(g) plurality of differential circuit connectors 90 formed below lower substrate 10;

(h) a differential electrode system consisting of a differential working electrode 110 and a differential counter electrode 120;

(i) a differential insulator 150;

(j) a differential porous membrane 160;

(k) a differential adhesive 170 covering the resulting structure with the exception of the porous membrane; and

(l) a differential protective membrane 220;

each of the differential component, established on symmetry with respect to the lower substrate 10, which functions in the same pattern as that of its corresponding one in the opposite module.

6. The biosensor as set forth in claim 1, wherein said biosensor is a differential face-to face type with a three-electrode system, comprising:

(a) a lower substrate 10;

(b) a plurality of spaced first circuit connector 9;

formed on the lower substrate 10,

(c) a counter electrode 12 and a reference electrode 13, both being coated on the circuit connectors 9 at a predetermined region;

(d) an insulator 15 covering the surface of the lower substrate 10 exception of the electrode system containing a counter electrode 12 and a reference electrode 13; and

plurality of spaced first circuit connectors 9 exposed on terminal region of the lower substrate 10;

(e) a porous membrane 16 located on the electrode system containing a counter electrode 12 and a reference electrode 13, having the same dimension as the exposed area of the electrode system with a counter electrode 12 and a reference electrode 13;

(f) an adhesive 17 deposited over the exception of the porous membrane 16, fixing the resulting structure;

(g) a working electrode 11 atop the porous membrane 16;

(h) a second circuit connector 9′

expanding to the working electrode 11;

(i) upper substrate 10′

covering with a second insulator 15′

while exposing exception of the working electrode 11;

(j) a plurality of differential circuit connectors 90 formed below the lower substrate 10;

(k) a differential counter electrode 120 and a differential reference electrode 130;

(l) a differential insulator 150 formed on the differential circuit connectors 90;

(m) a differential porous membrane 160;

(n) a differential adhesive 170 with the exception of the porous membrane 160;

(o) a differential working electrode 110 atop the differential porous membrane 160;

(p) a second differential circuit connector 90′

with the differential adhesive 170;

(q) a differential second insulator 150′

; and

(r) a differential upper substrate 100, each of the differential component, established on symmetry with respect to the lower substrate 10, which functions in the same pattern as that of its corresponding one in the opposite module.

7. The biosensor as set forth in claim 1, wherein said biosensor is a differential face-to-face type with a two-electrode system, comprising:

(a) a lower substrate 10;

(b) a plurality of spaced first circuit connector 9;

formed on the lower substrate 10,

(c) a counter electrode 12 and a reference electrode 13, both being coated on the circuit connectors 9 at a predetermined region;

(d) an insulator 15 covering the surface of the lower substrate 10 exception of the electrode 12; and

plurality of spaced first circuit connectors 9 exposed on terminal region of the lower substrate 10;

(e) a porous membrane 16 located on the electrode system containing a counter electrode 12, having the same dimension as the exposed area of the electrode system with a counter electrode;

(f) an adhesive 17 deposited over the lower substrate 10 with the exception of the porous membrane 16, fixing the resulting structure;

(g) a working electrode 11 atop the porous membrane 16;

(h) a second circuit connector 9′

expanding to the working electrode 11;

(i) upper substrate 10 covering with a second insulator 15′

while exposing exception of the working electrode 11;

(j) a plurality of differential circuit connectors 90 formed below the lower substrate 10;

(k) a differential counter electrode 120;

(l) a differential insulator 150 formed on the differential circuit connectors 90;

(m) a differential porous membrane 160;

(n) a differential adhesive 170 with the exception of the porous membrane 160;

(o) a differential working electrode 110 atop the differential porous membrane 160;

(p) a second differential circuit connector 90′

with the differential adhesive 170;

(q) a differential second insulator 150′

; and

(r) a differential upper substrate 100, each of the differential component, established on symmetry with respect to the lower substrate 10, which functions in the same pattern as that of its corresponding one in the opposite module.

8. The biosensor as set forth in claim 3, wherein said biosensor is a face-to-face type with a three-electrode system, comprising:

(a) a lower substrate 10;

(b) an electrodesystem formed on the lower substrate 10, consisting of a J-shaped working electrode connector strip 11′

with a working electrode 11 established thereon, a counter electrode connector strip 12′

, and a reference electrode connector strip 13′

with a reference electrode 13 established thereon;

(c) a porous membrane 16, either said porous membrane 16 or the working electrode 11 having an oxidase and an electron transfer mediator immobilized thereto;

(d) an adhesive 17 covered over the lower substrate 10 with the exception of the porous membrane 16;

(e) a Y-shaped circuit connector strip 20 with a counter electrode 12 on the branched line ends, and

(f) an upper substrate 10′

, fixed by the adhesive 17, having a hole of upper substrate 18 through which samples are introduced inside the biosensor.

9. The biosensor as set forth in claim 3, wherein said biosensor is a face-to-face type with a three-electrode system, comprising:

(a) a lower substrate 10;

(b) an electrode system formed on the lower substrate 10, consisting of a J-shaped working electrode connector strip 11′

with a working electrode 11 established thereon, a counter electrode connector strip 12′

, and a reference electrode connector strip 13′

with a reference electrode 13 established thereon;

(c) a porous membrane 16, either said porous membrane 16 or the working electrode 11 having an oxidase and an electron transfer mediator immobilized thereto;

(d) an adhesive 17 covered over the lower substrate 10 with the exception of the porous membrane 16;

(e) a Y-shaped circuit connector strip 20 with a counter electrode 12 on the branched line ends, and

(f) an upper substrate 10′

, fixed by the adhesive 17, having a hole of upper substrate 18; and

(g) a second upper substrate 10″

, fixed to the first upper substrate 10′

via an adhesive 17, having a sample inlet 14, which is formed in the direction traversing the electrode system, wherein the sample inlet has two opposite open ends through which samples are introduced to the biosensor.

10. The biosensor as set forth in claim 3, wherein said biosensor is a face-to-face type with a three-electrode system, comprising:

(a) a lower substrate 10;

(b) an electrode system formed on the lower substrate 10, consisting of a J-shaped working electrode connector strip 11′

with a working electrode 11 established thereon, a counter electrode connector strip 12′

, and a reference electrode connector strip 13′

with a reference electrode 13 established thereon;

(c) a porous membrane 16, either said porous membrane 16 or the working electrode 11 having an oxidase and an electron transfer mediator immobilized thereto;

(d) an adhesive 17 covered over the lower substrate 10 with the exception of the porous membrane 16;

(e) a Y-shaped circuit connector strip 20 with a counter electrode 12 on the branched line ends, and

(f) an upper substrate 10′

, via the adhesive 17, having a hole of upper substrate 18; and

(g) a second upper substrate 10″

with a second hole of upper substrate 18′

fixed to the first upper substrate 10′

via an adhesive 17, having a sample inlet 14 which is formed in the same direction as in the electrode system, extending from a mid point to an end of the second upper substrate 10″

, wherein the sample inlet has one open end through which samples are introduced to the biosensor.

11. The biosensor as set forth in claim 3, wherein said biosensor is a face-to-face type employing a two-electrode system, comprising:

(a) a lower substrate 10;

(b) an electrode system formed on the lower substrate 10, consisting of a Y-shaped working electrode connector strip 11′

with a working electrode 11 established thereon and a counter electrode connector strip 12′

the latter it is connected to one end portion of the circuit connector strip 20 on the upper substrate 10′

;

(c) a porous membrane 16, either said porous membrane 16 or the working electrode 11 having an oxidase and an electron transfer mediator immobilized thereto;

(d) an adhesive 17 covered over the lower substrate 10 with the exception of the porous membrane 16;

(e) a Y-shaped circuit connector strip 20 with a counter electrode 12 on the branched line ends, and

(f) an upper substrate 10′

, via the adhesive 17, having a hole of upper substrate 18; and

(g) a second upper substrate 101″

, having a sample inlet, fixed to the first upper substrate 10′

14 via an adhesive 17. Wherein said a sample inlet 14 is formed in the direction traversing the electrode system, wherein the sample inlet has two opposite open ends through which samples are introduced to the biosensor.

12. The biosensor as set forth in claim 3, wherein said biosensor is a face-to-face type with a two-electrode system, comprising:

(a) a lower substrate 10;

(b) an electrode system formed on the lower substrate 10, consisting of a Y-shaped working electrode connector strip 11′

with a working electrode 11 established thereon and a counter electrode connector strip 12′

established thereon;

(c) a porous membrane 16, either said porous membrane 16 or the working electrode 11 having an oxidase and an electron transfer mediator immobilized thereto;

(d) an adhesive 17 covered over the lower substrate 10 with the exception of the porous membrane 16;

(e) a Y-shaped circuit connector strip 20 with a counter electrode 12 on the branched line ends,

(f) an upper substrate 10′

, fixed by the adhesive 17, having a hole of upper substrate 18; and

(g) a second upper substrate 10″

with a second hole of upper substrate 18′

, having a sample inlet 14, fixed to the first upper substrate 10′

via an adhesive 17, wherein said a sample inlet 14 is formed in the same direction as in the electrode system, wherein the sample inlet has one open end through which samples are introduced to the biosensor.

13. The biosensor as set forth in claim 3, wherein said biosensor is a face-to-face type employing a two-electrode system, all of the electrodes being made of carbon ink only, comprising:

(a) a lower substrate 10;

(b) an electrode system formed on the lower substrate 10, consisting of a Y-shaped working electrode connector strip 11′

with a working electrode 11 established thereon and a counter electrode connector strip 12′

;

(c) a porous membrane 16, either said porous membrane 16 or the working electrode 11 having an oxidase and an electron transfer mediator immobilized thereto;

(d) an adhesive 17 covered over the lower substrate 10 with the exception of the porous membrane 16;

(e) a Y-shaped circuit connector strip 20 with a counter electrode 12 on the branched line ends,

(f) an upper substrate 10′

, formed on the adhesive 17, having a hole of upper substrate 18; and

(g) a second upper substrate 10″

, having a sample inlet 14, fixed to the first upper substrate 10′

via an adhesive 17, wherein said a sample inlet 14 is formed in the direction traversing the electrode system, wherein the sample inlet has two opposite open ends through which samples are introduced to the biosensor.

14. The biosensor as set forth in claim 3, wherein said biosensor is a face-to-face type employing a two-electrode system, characterized in that a sample inlet 14 is formed in an upper substrate 10 not in a second upper substrate 10″

and all electrodes are fabricated by carbon ink only, comprising;

(a) a lower substrate 10;

(b) an electrode system formed on the lower substrate 10, consisting of a Y-shaped working electrode connector strip 11′

with a working electrode 11 established thereon and a counter electrode connector strip 12′

, all of the electrodes coating carbon ink only;

(c) a porous membrane 16, formed on a predetermined area of the electrode system, covering the working electrode 11;

(d) an adhesive 17 covered over the lower substrate 10 with the exception of the porous membrane 16;

(e) a Y-shaped circuit connector strip 20 with a counter electrode 12 coated with carbon ink only,

(f) an upper substrate 10′

, fixed by the adhesive 17, having a sample inlet 14 extending in the lengthwise direction to one end of the upper substrate 10′

from a mid point and thus having an open end; and

(g) a second upper substrate 10″

, fixed to the first upper substrate 10′

via an adhesive 17, having a second hole of upper substrate 18′

.

15. The biosensor as set forth in claim 3, wherein said biosensor is a face-to-face type with a three-electrode system characterized by providing a circular space for a sample inlet. Comprising:

(a) a lower substrate 10;

(b) an electrode system formed on the lower substrate 10, consisting of a J-shaped working electrode connector strip 11′

with a working electrode 11 established thereon, a counter electrode connector strip 12′

, and a reference electrode connector strip 13′

with a reference electrode 13 established thereon;

(c) a porous membrane 16, either said porous membrane 16 or the working electrode 11 having an oxidase and an electron transfer mediator immobilized thereto;

(d) an adhesive 17 covered over the lower substrate 10 with the exception of the porous membrane 16;

(e) a Y-shaped circuit connector strip 20 with a counter electrode 12 on the branched line ends,

(f) an upper substrate 10′

, fixed by the adhesive 17, having a hole of upper substrate 18 through which samples are introduced inside the biosensor.

(g) a circular second upper substrate 10″

with a circular sample inlet 14, fixed to the first upper substrate 10′

via an adhesive 17.

16. The biosensor as set forth in claim 3, wherein said biosensor is a face-to-face type with a two-electrode system characterized by providing a circular space for a sample inlet. Comprising:

(a) a lower substrate 10;

(b) an electrode system formed on the lower substrate 10, consisting of a J-shaped working electrode connector strip 11′

with a working electrode 11 established thereon, a counter electrode connector strip 12′

, the latter it is connected to one end portion circuit connector strip 20 on the upper substrate 10′

;

(c) a porous membrane 16, either said porous membrane 16 or the working electrode 11 having an oxidase and an electron transfer mediator immobilized thereto;

(d) an adhesive 17 covered over the lower substrate 10 with the exception of the porous membrane 16;

(e) a Y-shaped circuit connector strip 20 with a counter electrode 12 on the branched line ends,

(f) an upper substrate 10′

, fixed by the adhesive 17, having a hole of upper substrate 18 through which samples are introduced inside the biosensor.

(g) a circular second upper substrate 10″

with a circular sample inlet 14, fixed to the first upper substrate 10′

via an adhesive 17.

17. The biosensor as set forth in claim 3, wherein said biosensor is a flat type adopting a three-electrode system, comprising:

(a) a lower substrate 10;

(b) a electrode system, formed on the lower substrate 10, consisting of a working electrode connector strip 11′

with a working electrode 11 established thereon, a counter electrode connector strip 12′

with a J-shaped counter electrode 12 established thereon, and a reference electrode connector strip 13′

with a reference electrode 13 established thereon;

(c) a porous membrane 16, formed on a predetermined area of the electrode system, covering the working electrode 11, and an adhesive 17 deposited over the intermediate membrane with the exception of the porous membrane 16;

(d) a upper substrate 10′

with a hole upper substrate 18 fixed to the lower substrate 10 via the adhesive 17; and

(e) a second upper substrate 10″

with a second hole of upper substrate 18′

, having a sample inlet, fixed to the first upper substrate 10′

14 via an adhesive 17, wherein said a sample inlet 14 is formed straightly extending from a mid point to one end of the second upper substrate 10″ and

is introduced samples to the biosensor.

18. The biosensor as set forth in claim 3, wherein said biosensor is a flat type employing a two-electrode system, comprising:

(a) a lower substrate 10;

(b) an electrode system, formed on the substrate 10, consisting of a working electrode connector strip 11′

with a working electrode 11 established thereon and a counter electrode connector strip 12′

with a J-shaped counter electrode 12 established thereon;

(c) a porous membrane 16, formed on a predetermined area of the electrode system, covering the working electrode 11, and an adhesive 17 deposited over the intermediate membrane with the exception of the porous membrane 16;

(d) a upper substrate 10′

with a hole upper substrate 18 fixed to the lower substrate 10 via the adhesive 17; and

(e) a second upper substrate 10″

with a second hole of upper substrate 18′

, having a sample inlet 14, fixed to the first upper substrate 10′

via an adhesive 17, wherein said a sample inlet 14 is formed straightly extending from a mid point to one end of the second upper substrate 10″ and

is introduced samples to the biosensor.

19. The biosensor as set forth in claim 3, wherein said biosensor is a flat type employing a three-electrode system, which has a porous membrane as a lower substrate, comprising:

(a) a lower substrate made of a porous membrane 16;

(b) an insulator 15 formed on one half portion of the lower substrate 16, an electrode system, formed on the insulator 15, consisting of a working electrode connector strip 11′

with a working electrode 11 established thereon, a counter electrode connector strip 12′

with a counter electrode 12 established thereon, and a reference electrode connector strip 13′

with a reference electrode 13 established thereon, and a pretreatment layer 19 adjacent to the insulator 15; and

(c) an upper substrate 10′

fixed onto the another half portion of the porous membrane 16 via an adhesive 17, having an sample inlet 14 which is formed in the direction traversing the electrode system and has two open ends. Through the sample inlet having two open ends, samples can be introduced from both the left and right ends of the biosensor adopting the three- electrode system.

20. The biosensor as set forth in claim 3, wherein said biosensor is a flat type employing a two-electrode system, which has a porous membrane as a lower substrate, comprising:

(a) a lower substrate made of a porous membrane 16;

(b) an insulator 15 formed on one half portion of the lower substrate 16, an electrode system, formed on the insulator 15, consisting of a working electrode connector strip 11′

with a working electrode 11 established thereon and a counter electrode connector strip 12′

with a counter electrode 12 established thereon and a pretreatment layer 19 adjacent to the insulator 15; and

(c) an upper substrate 10′

fixed onto the another half portion of the porous membrane 16 via an adhesive 17, having an sample inlet 14 which is formed in the direction traversing the electrode system and has two open ends. Through the sample inlet having two open ends, samples can be introduced from both the left and right ends of the biosensor adopting the three-electrode system.

21. The biosensor as set forth in claim 1, wherein the porous membrane 16 contains pores ranging from 5 to 20 μ

m in diameter.

22. The biosensor as set forth in claim 1, wherein the porous membrane 16 is selected from paper, a hydrophilic organic polymer or a hygroscopic ceramic polymer.

23. The biosensor as set forth in claim 22, wherein a hydrophilic organic polymer or a hygroscopic ceramic polymer is selected from the group consisting of nylon, hydrophilic polyester sulfone membranes, hydrophilic mixed cellulose esters, polytetrafluoroethylene membranes, polyvinylidine fluoride membranes, ion-selective membranes, glass fiber, polyester fiber or its modified fiber membranes.

24. The biosensor as set forth in claim 1, wherein the porous membrane 16 is preferable use of nitrocellulose paper, polyester fiber or its modified fiber membranes or similar filter paper.

25. The biosensor as set forth in claim 1, wherein the oxidase is selected from the group consisting of glucose oxidase, lactate oxidase, cholesterol oxidase, glutamate oxidase, horse radish peroxidase and alcohol oxidase.

26. The biosensor as set forth in claim 1, wherein the electron transfer mediator is selected from the group consisting of hexaamineruthenium (III) chloride, potassium ferricyanide, potassium ferrocyanide, dimethylferrocene (DMF), ferricinium, ferocene-monocarboxylic acid (FCOOH), 7,7,8,8-tetracyanoquinodimethane (TCNQ), tetrathiafulvalene (TTF), nickelocene (Nc), N-methylacidinium (NMA⁺), tetrathiatetracene (TTT), N-methylphenazinium (NMP⁺), hydroquinone, 3-dimethylaminobenzoic acid (MBTHDMAB), 3-methyl-2-benzothiozolinone hydrazone, 2-methoxy-4-allylphenol, 4-aminoantipyrine (AAP), dimethylaniline, 4-aminoantipyrene, 4-methoxynaphthol, 3,3′

,5,5′

-tetramethylbenzidine (TMB), 2,2-azino-di-[3-ethylbenzothizoline sulfonate], o-dianisidine, o-toluidine, 2,4-dichloro phenol, 4-aminophenazone, benzidine, prussian blue as mixed-valence compounds capable of forming redox couples.

27. The biosensor as set forth in claim 1, wherein the substrate is formed of ceramic, plastic, silicon, alumina glass plates, or polymeric materials.

28. The biosensor as set forth in claim 1, wherein the protective membrane 22 is formed of an organic polymer selected from the group consisting of polyester, polyvinyl chloride, and polycarbonate.

29. The biosensor as set forth in claim 1, wherein the electrode is fabricated in a direct chemical vacuum deposition process or in a plasma deposition process.

30. The biosensor as set forth in claim 3, wherein the sample inlet 14 is modified with a surfactant or by additives to improve the mobility of the whole blood sample through the porous membrane.

31. The biosensor as set forth in claim 3, wherein the sample inlet 14 is modified in morphology and size by applying grooves to the sample inlet or the hole.

32. The biosensor as set forth in claim 3, wherein the pretreatment layer 19 is provided with a sample inlet for removing various interfering materials, whereby the quantification of samples of interest can be improved in measurement convenience, accuracy and precision.