Electrochemical system for determining blood coagulation time

• US 6,352,630 B1
• Filed: 02/08/2000
• Issued: 03/05/2002
• Est. Priority Date: 02/23/1999
• Status: Expired due to Term

• Current Assignee: Asulab SA (The Swatch Group AG)
• Original Assignee: Asulab SA (The Swatch Group AG)
• Inventors: Frenkel, Erik Jan, Haeberli, André, Moresi, Anita
• Primary Examiner(s): Warden, Sr., Robert J.
• Assistant Examiner(s): OLSEN, KAJ K
• Application Number:
  US09/499,358
• Time in Patent Office: 756 Days
• Field of Search: 435/13, 422/73, 204/403, 205/775
• US Class Current: 204/403.02
• CPC Class Codes: C12Q 1/56 involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• CPC Class Codes: G01N 2333/974 Thrombin
• CPC Class Codes: G01N 33/4905 Determining clotting time of blood by chemical methods G01N33/86, C12Q1/54

Abstract

Electrochemical system for measuring a value representing the coagulation time of a drop of whole blood including:

an electrochemical sensor in the shape of a strip of small dimensions bearing at least a reference electrode and a working electrode on which a specific reagent is immobilized, including at least one chemical substrate one terminal link of which can be cut off by the thrombin enzyme to give charged groups (LG), and

a measuring apparatus intended to receive said sensor allowing a given voltage or current to be imposed across the electrodes of the sensor, the electric signal resulting from the migration of the charged groups to be processed, said signal to be correlated with a value representing the coagulation time, and said value to be displayed on a display panel.

The specific reagent preferably includes an oligopeptidic derivative as a substrate, a thromboplastin and a buffer medium.

Claims

1. An electrochemical system for measuring a value representing the coagulation time of a drop of whole blood wherein said system includes:

an electrochemical sensor in the shape of a strip bearing at least a reference electrode and a working electrode on which a specific reagent is immobilised on a measuring zone, the composition of said reagent incorporating at least a thromboplastin having a quantity of negative phospholipids greater than that of positive phospholipids, a buffer medium and an oligopeptidic substrate, one terminal link of which can be cut off by the thrombin enzyme to give charged groups (LG), and

a portable measuring apparatus dimensionally configured to receive the sensor and having an electronic circuit, which is supplied with power by a power source, allowing a variable or non variable electric current or a variable or non variable potential difference to be imposed across the electrodes of the sensor, said measuring apparatus having means for processing the electric signal resulting from the migration of the charged groups (LG), means for correlating said signal with a value representative of the coagulation time, and means for displaying said value on a display device of said measuring apparatus.

2. The measuring system according to claim 1, wherein the oligopeptidic substrate is a tripeptide having, as terminal amino-acid, arginine (Arg) linked to an amine function of a substituted amino-aryl group, said group being able to be cut off by thrombin to give the charged groups (LG).

3. The measuring system according to claim 2, wherein other amino acids of the tripeptide are selected from the group consisting of 2-aminobutyric acid (Abu), alanine (Ala), 3-cychlohexylalanine (Cha), 2-cyclohexylglycine (Chg), phenylalanine (Phe), pipecolic acid (Pip), proline (Pro), valine (Val) and glycine (Gly), said amino-acids being able to be in the L, D or DL form.

4. The measuring system according to claim 2, wherein the amino-aryl group (LG) of the oligopeptidic substrate is selected from the group consisting of aniline and quinolylamine derivatives substituted by one or more substituents selected from the group consisting of an halogen, an hydroxy, amino, nitro, alkyl, alkoxy, alkanoyl, anilino and aminophenyl radical.

5. The measuring system according to claim 2, wherein the first amino-acid of the oligopeptidic substrate further has a hydrogen of its amino function replaced by a protective group selected from the group consisting of Boc (ter-butoxycarbonyl), Tos (paratoluenesulfonic), t-Bups (ter-butylphenylsulfonyl), Mes (methylsulfonyl), Naps (2-naphtylsulfonyl), Bzo (benzoyl), Z (benzyloxycarbonyl), isopropylsulfonyl and camphosulfonyl acids.

6. The measuring system according to claim 5, wherein the oligopeptidic substrate is selected from the group consisting of:

Z-Gly-Pro-Arg-3-chloro-4-hydroxyanilide

Tos-Gly-Pro-Arg-3-chloro-4-hydroxyanilide

Boc-(D)-Chg-Gly-Arg-3-chloro-4-hydroxyanilide

H-(D)-Chg-Gly-Arg-3-chloro-4-hydroxyanilide

Z-Gly-Pro-Arg-2-chloro-4-hydroxyanilide

and compatible mineral or organic salts of said oligopeptidic substrate.

7. The measuring system according to claim 6, wherein an oligopeptidic substrate salt is formed with hydrochloric acid (HCl), acetic acid or trifluoroacetic acid (TFA).

8. The measuring system according to claim 1, wherein the level of the negative phospholipids is greater than 65%.

9. The measuring system according to claim 1, wherein the specific reagent of the sensor further includes in its composition a calcium salt such as CaCl₂.

10. The measuring system according to claim 1, wherein the buffer of the specific reagent of the sensor is the HEPES buffer.

11. The measuring system according to claim 1, wherein the specific reagent of the sensor is first prepared in liquid or pasty form, applied onto the working electrode with a pipette or by silk screen process, and then brought to the dry state by lyophilisation.

12. The measuring system according to claim 1, wherein the power source of the portable measuring apparatus is an autonomous power source including a battery.

13. The measuring system according to claim 1, wherein the portable measuring apparatus further includes means for bringing the measuring zone to a determined temperature and keeping it at that temperature.

14. An electrochemical system for measuring a value representing the coagulation time of a drop of whole blood wherein said system includes:

an electrochemical sensor in the shape of a strip bearing at least a reference electrode and a working electrode on which a specific reagent is immobilised, the composition of said reagent incorporating at least one chemical substrate, one terminal link of which can be cut off by the thrombin enzyme to give charged groups (LG), and

a measuring apparatus dimensionally configured to receive the sensor and whose electronic circuit, which is supplied with power by a power source, allowing a variable or non variable electric current or a variable or non variable potential difference to be imposed across the electrodes of the sensor, said measuring apparatus having means for processing the electric signal resulting from the migration of the charged groups (LG), means for correlating said signal with a value representative of the coagulation time, and means for displaying said value on a display device of said measuring apparatus;

wherein the chemical substrate of the specific reagent is an oligopeptidic derivative;

wherein the oligopeptidic derivative has, as terminal amino-acid, arginine (Arg) linked to an amine function of a substituted amino-aryl group, said group being able to be cut off by thrombin to give the charged groups (LG); and

wherein the oligopeptidic substrate is selected from the group consisting of;

Z-Gly-Pro-Arg-3-chloro-4-hydroxyanilide

Tos-Gly-Pro-Arg-3-chloro-4-hydroxyanilide

Boc-(D)-Chg-Gly-Arg-3-chloro-4-hydroxyanilide

H-(D)-Chg-Gly-Arg-3-chloro-4-hydroxyanilide

Z-Gly-Pro-Arg-2-chloro-4-hydroxyanilide

and compatible mineral or organic salts of said oligopeptidic substrate.

15. The measuring system according to claim 14, wherein an oligopeptidic substrate salt is formed with hydrochloric acid (HCl), acetic acid or trifluoroacetic acid (TFA).