Cholesterol Sensor
A method for the determination of the amount of cholesterol in lipoproteins other than high density lipoproteins in a lipoprotein-containing sample, said method comprising (a) electrochemically determining the amount of cholesterol bound to high density lipoproteins in the sample, (b) electrochemically determining the total amount of cholesterol in the sample, and subtracting the result of (a) from the result of (b).
1 . A method for the determination of the amount of cholesterol in lipoproteins other than high density lipoproteins in a lipoprotein-containing sample, said method comprising (a) electrochemically determining the amount of cholesterol bound to high density lipoproteins in the sample, (b) electrochemically determining the total amount of cholesterol in the sample, and subtracting the result of (a) from the result of (b).
2 . A method according to claim 1 , wherein step (a) comprises reacting the sample with a first series of reagents comprising
(a1) a cholesterol ester hydrolysing reagent;
(a2) cholesterol dehydrogenase;
(a3) a coenzyme;
(a4) a redox agent capable of being oxidised or reduced to form a product; and
(a5) a surfactant which selectively breaks down high density lipoproteins,
and electrochemically detecting the amount of product formed.
3 . A method according to claim 2 , wherein the surfactant (a5) has a differentiation for HDL over LDL given by the equation (i) below of at least 50%:
Differentiation
(
%
)
=
G
HDL
-
G
LDL
G
HDL
×
100
wherein
G
HDL
is
measured
(
HDLcholesterol
)
known
[
HDLcholesterol
]
,
and
G
LDL
is
measured
(
LDLcholesterol
)
known
[
LDLcholesterol
]
.
(
i
)
wherein measured (HDL cholesterol) or measured (LDL cholesterol) is a measured value relating to the concentration of HDL or LDL respectively.
4 . A method according to claim 2 , wherein the surfactant (a5) is a sucrose ester, maltoside, hydroxyethylglucamide derivative or N-methyl-N-acyl glucamine derivative.
5 . A method according to claim 2 , wherein the surfactant (a5) is of formula CH 3 (CH 2 ) 13 (OCH 2 CH 2 ) 33.5 OH, a hydroxyethyl glucamide derivative or sucrose monocaprate.
6 . A method according to claim 1 , wherein step (b) comprises reacting the sample with a second series of reagents comprising
(b1) a cholesterol ester hydrolysing reagent;
(b2) cholesterol dehydrogenase;
(b3) a coenzyme;
(b4) a redox agent capable of being oxidised or reduced to form a product; and optionally
(b5) a surfactant,
and electrochemically detecting the amount of product formed.
7 . A method according to claim 6 , wherein the surfactant (b5) comprises one or more bile acid derivatives or salts thereof.
8 . A method according to claim 6 , wherein the sample is reacted substantially simultaneously with the first series of reagents and the second series of reagents.
9 . A method according to claim 2 , wherein step (a) comprises reacting the sample with the first series of reagents and electrochemically detecting the product formed at a time when the high density lipoproteins preferentially react; and step (b) comprises continuing the reaction between the sample and the first series of reagents and electrochemically detecting the product formed at a time when all lipoproteins react.
10 . A method according to claim 2 , wherein the first and/or second series of reagents additionally comprises a reductase.
11 . A method according to claim 1 , wherein the lipoprotein containing sample is whole blood and wherein the method additionally comprises the step of filtering the sample to remove red blood cells.
12 . A method according to claim 1 , wherein the method is completed within a total period of no more than 3 minutes.
13 . A kit for the determination of the amount of cholesterol in lipoproteins other than high density lipoproteins in a lipoprotein-containing sample, the kit comprising
a first electrochemical cell having a working electrode, a reference or pseudo reference electrode and optionally a separate counter electrode;
a first series of reagents comprising
(a1) a cholesterol ester hydrolysing reagent;
(a2) cholesterol dehydrogenase;
(a3) a coenzyme;
(a4) a redox agent capable of being oxidised or reduced to form a product; and
(a5) a surfactant which selectively breaks down high density lipoproteins, said first series of reagents being associated with said first electrochemical cell;
a second electrochemical cell having a working electrode, a reference or pseudo-reference electrode and optionally a separate counter electrode;
a second series of reagents comprising
(b1) a cholesterol ester hydrolysing reagent;
(b2) cholesterol dehydrogenase;
(b3) a coenzyme;
(b4) a redox agent capable of being oxidised or reduced to form a product; and optionally
(b5) a surfactant, said second series of reagents being associated with said second electrochemical cell;
a power supply for applying a potential across each cell;
a measuring instrument for measuring the resulting electrochemical response of each cell; and
a calculator for calculating the amount of cholesterol in lipoproteins other than high density lipoproteins.
14 . A kit according to claim 13 , wherein the first series of reagents is present in the form of a single first reagent mixture, and the second series of reagents is present in the form of a single second reagent mixture.
15 . A kit according to claim 14 , wherein each reagent mixture is in dried form.
16 . A method of operating a kit, which kit is for the determination of the amount of cholesterol in lipoproteins other than high density lipoproteins in a lipoprotein-containing sample the kit comprising
a first electrochemical cell having a working electrode, a reference or pseudo reference electrode and optionally a separate counter electrode;
a first series of reagents comprising
(a1) a cholesterol ester hydrolysing reagent;
(a2) cholesterol dehydrogenase;
(a3) a coenzyme;
(a4) a redox agent capable of being oxidised or reduced to form a product; and
(a5) a surfactant which selectively breaks down high density lipoproteins, said first series of reagents being associated with said first electrochemical cell;
a second electrochemical cell having a working electrode, a reference or pseudo reference electrode and optionally a separate counter electrode;
a second series of reagents comprising
(b1) a cholesterol ester hydrolysing reagent;
(b2) cholesterol dehydrogenase;
(b3) a coenzyme;
(b4) a redox agent capable of being oxidised or reduced to form a product; and optionally
(b5) a surfactant,
said second series of reagents being associated with said second electrochemical cell;
a power supply for applying a potential across each cell;
a measuring instrument for measuring the resulting electrochemical response of each cell; and
a calculator for calculating the amount of cholesterol in lipoproteins other than high density lipoproteins,
said method comprising
(i) contacting a sample with the first series of reagents, such that the resulting mixture of sample and first series of reagents is in contact with the working electrode of the first electrochemical cell;
(ii) contacting the sample with the second series of reagents, such that the resulting mixture of sample and second series of reagents is in contact with the working electrode of the second electrochemical cell;
(iii) applying a potential across each electrochemical cell;
(iv) electrochemically detecting the amount of product formed in each cell by measuring the resulting electrochemical response; and
(v) calculating the amount of cholesterol bound to non-HDL lipoproteins in the sample.
17 . A method according to claim 16 , wherein steps (i) and (ii) are carried out substantially simultaneously.
18 . A method according to claim 16 wherein step (v) is completed within a period of up to 3 minutes from the time at which the sample is contacted with the first and/or second series of reagents.