US2008268421A1PendingUtilityA1
Cholesterol Sensor
Assignee: WALLACE-DAVIS EMMA NAOMI KATHLEENPriority: Dec 21, 2005Filed: Dec 21, 2006Published: Oct 30, 2008
Est. expiryDec 21, 2025(expired)· nominal 20-yr term from priority
C12Q 1/005C12Q 1/004
47
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Claims
Abstract
A method for the determination of the amount of cholesterol in a sample is provided. The method typically provides a breakdown of the HDL and LDL cholesterol contents of the sample.
Claims
exact text as granted — not AI-modified1 . An electrochemical method for the determination of the total amount of cholesterol in a sample, said method comprising reacting the sample with a first series of reagents comprising:
(a1) a surfactant comprising one or more bile acid derivatives or salts thereof; (b1) a cholesterol ester hydrolysing reagent; (c1) cholesterol dehydrogenase; (d1) a coenzyme; and (e1) a redox agent capable of being oxidised or reduced to form a product,
and electrochemically detecting the amount of product formed.
2 . A method according to claim 1 wherein the cholesterol ester hydrolysing reagent (b1) is cholesterol esterase or a lipase.
3 . A method according to claim 1 , wherein the sample is further reacted with a second series of reagents to determine the triglyceride content of the sample, the second series of reagents comprising:
(a2) a surfactant comprising one or more bile acid derivatives or salts thereof; (b2) a triglyceride hydrolysing reagent; (c2) glycerol dehydrogenase; (d2) a coenzyme; and (e2) a redox agent capable of being oxidised or reduced to form a product,
and electrochemically detecting the amount of product formed.
4 . A method according to claim 1 , wherein the first series of reagents additionally comprises a reductase.
5 . A method according to claim 4 , wherein the reductase is diaphorase or putidaredoxin reductase.
6 . A method according to claim 1 , wherein the coenzyme (d1) is TNAD.
7 . A method according to claim 1 , wherein the redox agent (e1) is Fe(CN) 6 3− , Ru(NH 3 ) 6 3+ , Ru(acac) 2 (Py-3-CO 2 H)(Py-3-CO 2 ), or ferrocenium monocarboxylic acid (FMCA).
8 . A method according to claim 1 , wherein the sample is further reacted with a third series of reagents to determine the content of cholesterol in the sample which is bound to high density lipoproteins.
9 . A method according to claim 27 , which further comprises determining the content of cholesterol in the sample which is bound to low density lipoproteins ([LDL]) using the following equation:
[
L
D
L
]
=
[
Total
cholesterol
]
-
[
H
D
L
]
-
[
Triglyceride
]
2.17
wherein [Total cholesterol] is the total concentration of cholesterol in the sample, [HDL] is the concentration of cholesterol bound to high density lipoprotein particles and [Triglyceride] is the triglyceride concentration of the sample, the concentrations being measured in mM.
10 . A method according to claim 27 , wherein the sample is reacted with the first, second and third series of reagents substantially simultaneously.
11 . A method according to claim 1 , wherein the sample is whole blood and wherein the method additionally comprises the step of filtering the sample to remove red blood cells.
12 . A first reagent mixture for use in an electrochemical method for the determination of the total amount of cholesterol in a sample, the first reagent mixture comprising:
(a1) a surfactant comprising one or more bile acid derivatives or salts thereof; (b1) a cholesterol ester hydrolysing reagent; (c1) cholesterol dehydrogenase; (d1) a coenzyme; (e1) a redox agent capable of being oxidised or reduced to form a product; and optionally (f1) a reductase.
13 . A second reagent mixture for use in an electrochemical method for the determination of the amount of triglyceride in a sample, the second reagent mixture comprising
(a2) a surfactant comprising one or more bile acid derivatives or salts thereof; (b2) a triglyceride hydrolysing reagent; (c2) glycerol dehydrogenase; (d2) a coenzyme; (e2) a redox agent capable of being oxidised or reduced to form a product; and optionally (f2) a reductase.
14 . A kit for measuring the total cholesterol content of a sample, the kit comprising
a first and optionally further electrochemical cells, each cell having a working electrode, a reference or pseudo reference electrode and optionally a separate counter electrode; a first series of reagents as defined in claim 1 , said first series of reagents being associated with said first electrochemical cell; a power supply for applying a potential across the or each cell; and a measuring instrument for measuring the resulting electrochemical response of the or each cell.
15 . A kit according to claim 14 for measuring the total cholesterol and triglyceride content of a sample, which kit comprises a second electrochemical cell and a second series of reagents associated therewith, the second series of reagents comprising:
(a2) a surfactant comprising one or more bile acid derivatives or salts thereof; (b2) a triglyceride hydrolysing reagent; (c2) glycerol dehydrogenase; (d2) a coenzyme; and (e2) a redox agent capable of being oxidised or reduced to form a product, and electrochemically detecting the amount of product formed.
16 . A kit according to claim 14 , which comprises a third electrochemical cell and a third series of reagents associated therewith, wherein the third series of reagents is for the determination of the content of cholesterol in the sample which is bound to high density lipoproteins.
17 . A kit according to claim 28 , which further comprises a fourth control electrochemical cell.
18 . A kit according to claim 28 , which further comprises a calculator for calculating the content of cholesterol in a sample which is bound to low density lipoproteins ([LDL]) using the following equation:
[
L
D
L
]
=
[
Total
cholesterol
]
-
[
H
D
L
]
-
[
Triglyceride
]
2.17
wherein [Total cholesterol] is the total concentration of cholesterol in the sample, [HDL] is the concentration of cholesterol bound to high density lipoproteins and [Triglyceride] is the triglyceride concentration of the sample, the concentrations being measured in mM.
19 . A kit according to claim 14 , wherein the working electrode of the or each electrochemical cell is a microelectrode having at least one dimension of less than 50 μm.
20 . A kit according to claim 14 , wherein the or each electrochemical cell is in the form of a receptacle or partial receptacle, the working electrode is in a wall of the receptacle or partial receptacle and the first series of reagents is provided as a reagent mixture in dried form which is at least partly contained within the receptacle or partial receptacle.
21 . A kit according to claim 17 comprising a strip having at least four receptacles or partial receptacles formed therein, each receptacle or partial receptacle having a first open part in a first surface of the strip to enable a sample to enter the receptacle or partial receptacle,
wherein the working electrode of each electrochemical cell is in a wall of the receptacle or partial receptacle, and wherein the reference or pseudo reference electrode of each electrochemical cell comprises a reference or pseudo reference electrode layer formed on at least a part of the first surface of the strip, and wherein one receptacle or partial receptacle contains the first series of reagents, a second receptacle or partial receptacle contains the second series of reagents and a third receptacle or partial receptacle contains the third series of reagents.
22 . (canceled)
23 . A method of operating a kit as defined in claim 14 , said method comprising
(i) contacting a sample with the reagents of the or each electrochemical cell; (ii) applying a potential across the or each electrochemical cell; and (iii) electrochemically detecting the amount of product formed in the or each electrochemical cell by measuring the resulting electrochemical response.
24 . An electrochemical method for the determination of the amount of triglyceride in a sample, said method comprising reacting the sample with a second series of reagents comprising
(a2) a surfactant comprising one or more bile acid derivatives or salts thereof; (b2) a triglyceride hydrolysing reagent; (c2) glycerol dehydrogenase; (d2) a coenzyme; (e2) a redox agent capable of being oxidised or reduced to form a product; and optionally (f2) a reductase, and electrochemically detecting the amount of product formed.
25 . A process for breaking down the lipoprotein structure of a sample comprising lipoproteins by using one or more bile acid derivatives or salts thereof, in an electrochemical method for the determination of the total amount of cholesterol in a sample, said method comprising reacting the sample with a first series of reagents comprising
(a1) said one or more bile acid derivatives or salts thereof; (b1) a cholesterol ester hydrolysing reagent; (c1) cholesterol dehydrogenase; (d1) a coenzyme; and (e1) a redox agent capable of being oxidised or reduced to form a product, and optionally (f1) a reductase, and electrochemically detecting the amount of product formed.
26 . A process for breaking down the lipoprotein structure of a sample comprising lipoproteins by using one or more bile acid derivatives or salts thereof, in an electrochemical method for the determination of the amount of triglyceride in a sample, said method comprising reacting the sample with a second series of reagents comprising
(a2) said one or more bile acid derivatives or salts thereof; (b2) a triglyceride hydrolysing reagent; (c2) glycerol dehydrogenase; (d2) a coenzyme; (e2) a redox agent capable of being oxidised or reduced to form a product; and optionally (f2) a reductase, and electrochemically detecting the amount of product formed.
27 . A method according to claim 3 , wherein the sample is further reacted with a third series of reagents to determine the content of cholesterol in the sample which is bound to high density lipoproteins.
28 . A kit according to claim 15 , which comprises a third electrochemical cell and a third series of reagents associated therewith, wherein the third series of reagents is for the determination of the content of cholesterol in the sample which is bound to high density lipoproteins.Join the waitlist — get patent alerts
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