HK1128119A - Lipovrotein senso - Google Patents

Description

Lipoprotein sensor

The invention relates to the use of glycol ethers in sensors. In particular, the present invention relates to the use of glycol ethers in biosensors that selectively solubilize Low Density Lipoproteins (LDL) in cholesterol with minimal interaction with the high density lipoproteins in cholesterol, thereby allowing the detection of LDL.

Cholesterol plays an important role in normal body function. It acts on the development of cell tissues, regeneration of cell membranes, hormones, and provides other functions. However, high levels of cholesterol in the blood increase the risk of coronary heart disease, which can lead to heart attacks. In addition, it is known to be associated with increased risk of stroke. Patients with high levels of blood cholesterol are considered to have hypercholesterolemia.

There are two major sources of cholesterol in the body. The first major source is from the body itself. Another major source is from foods such as meat, poultry, fish and dairy products. Foods high in saturated fat promote the body to increase cholesterol production.

Cholesterol is transmitted to and from cells by specific carriers, known as lipoproteins. Because it is insoluble in blood. There are two main types of lipoproteins. They are Low Density Lipoproteins (LDL) and High Density Lipoproteins (HDL). LDL is known to be a "bad" cholesterol carrier form; HDL is known to be a "good" cholesterol carrier. LDL cholesterol tends to accumulate in the inner walls of arteries, causing plaque deposits that can block the arteries, causing an increased risk of heart attack or stroke. The ideal LDL cholesterol level in the blood is about 100 mg/dl. Higher levels (above 160mg/dl) indicate increased risk of heart disease.

HDL cholesterol is thought to protect the body from the increased risk of heart disease. HDL is thought to transport cholesterol out of the arteries and back to the liver. In addition, HDL can also remove excess cholesterol from plaque deposits already present in the arteries.

Therefore, efforts have been made to develop sensors capable of discriminating the amounts of LDL cholesterol and HDL cholesterol in blood.

Traditionally, differential ultracentrifugation was used to determine the amount of cholesterol in low density lipoproteins. However, this requires special equipment and can take a long time to obtain the required measurements.

More recently, sensors have been developed that are easier to use and provide more reliable results. Such sensors are generally referred to as biosensors.

Biosensors are analytical tools that combine biochemical recognition components or sensing elements with physical transducers. They have wide application in such diverse areas as personal health monitoring, environmental screening and monitoring, bioprocess monitoring, and within the food and beverage industry.

The biosensing element may be an enzyme, an antibody, a DNA sequence or even a microorganism. The biochemical component is used to selectively catalyze a reaction or promote binding. The selectivity of the biochemical recognition effect allows the operation of biosensors in complex sample matrices, i.e. body fluids. The transducer converts biochemical action into a measurable signal, thereby providing a means for detecting it. Measurable effects range from changes in the spectrum due to the production or consumption of enzyme reaction products/substrates to changes in mass upon biochemical complexation. Typically, transducers take many forms and they are indicative of the physicochemical parameter to be measured. Thus, the transducer may be optically based, measuring for example optical absorption, fluorescence or changes in refractive index. It may be mass-based, measuring the change in mass associated with a binding reaction of biological origin. In addition, it may be thermal-based (measuring changes in enthalpy (heat)) or impedance-based (measuring changes in electrical properties) that accompany analyte/bio-recognition layer interactions, or electrochemical-based.

Biosensors offer the convenience and ease of distributed measurement, i.e., the potential to enable the assay to fulfill a concern or care purpose. Biosensor devices that are properly designed and manufactured can be conveniently mass produced. However, there are several limitations to the use of biosensors. These limitations include the vulnerability of the converter to fouling and interference.

Enzyme-based biosensors are widely used to detect analytes in clinical, environmental, agricultural and biotechnological applications. Analytes that can be measured in clinical testing of fluids in the human body include, for example, glucose, lactate, cholesterol, bilirubin, and amino acids. The levels of these analytes in biological fluids, such as blood, are important for the diagnosis and monitoring of diseases.

Sensors commonly used in enzyme-based systems are provided as devices that are saleable for care purposes or over the counter. They can be used to test fresh unmodified full finger bleed samples to determine the concentration of total cholesterol, triglycerides, HDL and LDL within, for example, 1-2 minutes after the sample is added to the device (note that this time is not fixed and can vary significantly). These 4 parameters, combined, have been demonstrated clinically to provide a very good indication of the risk of heart disease in adults. It is well known that high cholesterol is asymptomatic. It is therefore recommended that each adult should be tested to assess their risk. If the risk is found to be high, it can be significantly reduced by proper diet control alone, or in combination with therapeutic drugs.

In one example of such an enzyme-based biosensor, an electrochemical assay is used to detect the analyte in question. This use is caused by a change in the oxidation state of a mediator which interacts with an enzyme which reacts with the analyte to be determined. The oxidation state of the mediator is selected so that it is only in a state that will interact with the enzyme upon addition of the substrate. The analyte reacts with the mediator by the enzyme. This causes the mediator to be oxidised or reduced (according to the enzymatic reaction) and this change in mediator level can be measured by determining an electrochemical signal, for example the current generated at a given voltage.

Conventional microelectrodes can be usedTypically with a working microelectrode and a reference electrode. The working electrode is typically composed of palladium, platinum, gold, or carbon. The counter electrode is typically carbon, Ag/AgCl, Ag/Ag₂SO₄Palladium, gold, platinum, Cu/CuSO₄、Hg/HgO、Hg/HgCl₂、Hg/HgSO₄Or Zn/ZnSO₄。

The working electrode may be located in a well of a container forming the microelectrode. Examples of microelectrodes that can be used therein are those disclosed in WO03/097860, the entire contents of which are hereby incorporated by reference.

The prior art teaches a number of methods for detecting LDL cholesterol in a sample, such as blood, serum or plasma. Many of these prior art methods for detecting cholesterol concentrations are based on the evaluation of various properties, such as color changes.

EP 1434054, WO 03/102596 and JP 2004-354284 disclose biosensors using polyethylene glycol ethers. Us patent No. 6762062 discloses a method for determining cholesterol in low density lipoproteins. The method is based on measuring the total cholesterol level in the sample and the cholesterol level in the non-LDL fraction (HDL, VLDL and chylomicrons). The amount of LDL cholesterol can then be determined by simply subtracting one amount from the other. Us patent No. 6342364 and JP 2001-343348 also disclose LDL detection systems based on the use of electrochemical cells.

It would therefore be advantageous to have a detection system that is simple to use but produces consistent and reliable results and does not require a change in color as part of the detection method.

According to a first aspect of the present invention there is provided a biosensor comprising a substrate containing a biochemical analyte, an enzyme system, a low molecular weight glycol ether and a detection means.

Typically, the substrate is a biological fluid, such as blood or plasma. The biochemical analyte determined from the biological fluid may be a lipoprotein, typically a low density lipoprotein.

The enzyme system may comprise a cholesterol enzyme, such as cholesterol esterase, cholesterol oxidase or cholesterol dehydrogenase.

The low molecular weight glycol ether may be selected from the group of linear or branched alkylene glycol groups having 1-4 repetitions, typically the alkylene group is 1, 2-ethylene, 1, 2-propylene and its isomers, butylene and its isomers, pentylene (pentylene) and its isomers, or a combination thereof. The glycol ethers being substituted by alkyl radicals, e.g. C₁-C₅Alkyl substitution. The low molecular weight glycol ether may be selected from 2-methoxyethanol, tripropylene glycol methyl ether, diethylene glycol propyl ether, diethylene glycol butyl ether, diethylene glycol pentyl ether, 1-methoxy-2-propanol, dipropylene glycol butyl ether, tripropylene glycol butyl ether, glycerol ethoxylate-co-propoxylate triol, neopentyl glycol ethoxylate, propoxyethol, triethylene glycol methyl ether, propylene glycol propyl ether, 1-tert-butoxy-2-propanol, dipropylene glycol propyl ether, tripropylene glycol propyl ether or dipropylene glycol tert-butyl ether.

The biosensor may further comprise an aqueous buffer solution. The buffer solution typically has a pH of 5-10. More preferably, the pH may range from 7 to 10.

The ionic strength or salt strength of the biosensor solution can be increased in order to improve selectivity for low density lipoproteins. The ionic strength may be increased by the addition of a salt selected from the group consisting of: potassium chloride, magnesium sulfate, ruthenium hexamine chloride, sodium chloride, calcium chloride, magnesium chloride, lanthanum chloride, sodium sulfate, or magnesium sulfate.

The detection means may be in the form of an electrochemical cell.

According to a second aspect of the present invention there is provided a detection system for measuring the amount of a biochemical analyte in a sample, comprising the steps of:

a) providing a mixture of a solution of a low molecular weight glycol ether and an enzyme mixture;

b) adding a solution of a sample to be detected;

c) incubating the resulting mixture under conditions that produce a measurable signal change;

d) measuring the resulting change; and

e) the amount of analyte or the difference between HDL and LDL is determined in the raw sample using a calibration curve.

The analyte may be a low density lipoprotein.

Typically, the measurable signal is an electrochemical signal, a colorimetric signal, a thermal signal, a piezoelectric signal, or a spectroscopic signal.

The biological analytes and reagents may be dried prior to use. The analytes and reagents may be freeze-dried.

According to a third aspect of the present invention there is provided the use of a low molecular weight glycol ether for solubilising a biochemical analyte.

The low molecular weight glycol ether may be selected from the group of straight or branched chain alkylene glycol groups having 1-4 repetitions, typically the alkylene group is 1, 2-ethylene, 1, 2-propylene and its isomers, butylene and its isomers, pentylene and its isomers, or combinations thereof. The glycol ethers being substituted by alkyl radicals, e.g. C₁-C₅Alkyl substitution. The low molecular weight glycol ether may be selected from 2-methoxyethanol, tripropylene glycol methyl ether, diethylene glycol propyl ether, diethylene glycol butyl ether, diethylene glycol pentyl ether, 1-methoxy-2-propanol, dipropylene glycol butyl ether, tripropylene glycol butyl ether, glycerol ethoxylate-co-propoxylate triol, neopentyl glycol ethoxylate, propoxyethanol, triethylene glycol methyl ether, propylene glycol propyl ether, 1-tert-butoxy-2-propanol, dipropylene glycol propyl ether, tripropylene glycol propyl ether or dipropylene glycol tert-butyl ether.

Glycol ethers may be used to solubilize lipoproteins, such as low density lipoprotein cholesterol.

In a fourth aspect of the invention, the ionic strength of the solution aids in the differentiation obtained between HDL and LDL cholesterol. It has been found that a change in the ionic strength or salt concentration of the liquid affects the relative extent of the reactions of the two cholesterols. Thus, there is provided the use of a salt for increasing the ionic strength or salt concentration of a solution containing low density lipoprotein, high density lipoprotein and glycol ether, wherein an increase in the ionic strength of the solution modulates the relative solubility of the low density lipoprotein and high density lipoprotein.

Typically, the use of the salt to increase the ionic strength or salt concentration increases the solubility of low density lipoproteins relative to high density lipoproteins.

The ionic strength or salt concentration of the solution can be controlled by the added salt, and in the examples potassium chloride, magnesium sulfate or ruthenium hexamine chloride is used to change the ionic strength or salt concentration of the solution. However, other salts may be used, such as potassium chloride, magnesium sulfate, ruthenium hexamine chloride, sodium chloride, calcium chloride, magnesium chloride, lanthanum chloride, sodium sulfate, or magnesium sulfate.

The following definitions, as used herein, define the terms:

the term "diol" refers to a dihydric alcohol. The term "glycol ether" refers to a monoalkyl ether of a dihydric or trihydric alcohol.

The term "alkyl" includes straight or branched chain saturated aliphatic hydrocarbons. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, and the like. Unless otherwise indicated, the term "alkyl" includes alkyl and cycloalkyl groups.

A "biological fluid" is any body fluid or body fluid derivative in which an analyte can be measured, for example, blood, urine, interstitial fluid, plasma, dermal fluid, sweat, and tears.

An "electrochemical sensor" is a device configured to detect the presence or measure the concentration or amount of an analyte in a sample via an electrochemical oxidation or reduction reaction.

A "redox mediator" is an electron transfer reagent used to transport electrons between an analyte or an enzyme for analyte reduction or an enzyme for analyte oxidation, a cofactor or other redox active species and an electrode, either directly or via one or more additional electron transfer reagents.

Unless otherwise indicated, the term "reference electrode" includes both: a) the reference electrode and b) may also function as a reference electrode for the counter electrode (i.e., counter/reference electrode).

Unless otherwise indicated, the term "counter electrode" includes both: a) a counter electrode and b) a counter electrode that can also function as a reference electrode (i.e., a counter-reference electrode).

The term "measurable signal" means a signal that can be readily measured, such as an electrical current, voltage, fluorescence, absorption spectrum, luminescence, light scattering, NMR, IR, mass spectrometry, heat exchange, or piezoelectric change.

The term "biochemical analyte" includes any measurable chemical or biochemical substance that may be present in a biological fluid, and also includes any enzyme, antibody, DNA sequence, or microorganism.

Known biosensors that can be used according to the invention may consist of, for example, a strip with 4 reagent wells and 1 common reference; wherein each aperture has its own microstrip working electrode, such as a tubular microstrip electrode. The sensing component of the strip is provided by drying a different, in particular formulated, reagent comprising at least one enzyme and a mediator which interacts with a specific analyte in the sample in each well. Since potentially different reagents can be added and dried to each well, it is clear that multi-analyte testing can be accomplished with a single sample. The number of wells, and thus the number of specific tests, may vary, for example sensors using 1-6 wells may be used.

Conventional microelectrodes, typically having a working microelectrode and a reference electrode, may be used. Worker's toolThe working electrode is usually composed of palladium, platinum, gold or carbon. The counter electrode is typically carbon, Ag/AgCl, Ag/Ag₂SO₄Palladium, gold, platinum, Cu/CuSO₄、Hg/HgO、Hg/HgCl₂、Hg/HgSO₄Or Zn/ZnSO₄。

In a preferred microelectrode, the working electrode is located in the container forming said microelectrode. Examples of microelectrodes that can be used according to the invention are those disclosed in WO 2003097860.

Embodiments of the invention will now be described by way of example only with reference to the following drawings, in which:

FIGS. 1 and 2 illustrate the results obtained for the selective solubilization of LDL in preference to HDL when using diethylene glycol monopentyl ether (example 1).

FIGS. 3 and 4 illustrate the results obtained for the selective solubilization of LDL in preference to HDL when using diethylene glycol monobutyl ether (example 1).

Figure 5 shows the results from example 2. (where E2C4 is diethylene glycol butyl ether). The gradient at each time point was used to calculate the% difference obtained from the measurement of LDL and HDL.

Figure 6 shows the results from example 3. The gradient at each time point was used to calculate the% difference obtained from the measurement of LDL and HDL.

Figure 7 shows the results from example 4. The gradient at each time point was used to calculate the% difference obtained from the measurement of LDL and HDL.

Figure 8 shows the results from example 5. The gradient at the first time point was used to calculate the% difference obtained between the measurement of LDL and HDL.

Figure 9 shows the results from example 6. The gradient at each time point was used to calculate the% difference obtained between the measurement of LDL and HDL.

Figure 10 shows the results from example 7. The gradient at each time point was used to calculate the% difference obtained between the measurement of LDL and HDL.

Fig. 11a-d show the results from example 8 for the first time point 0 seconds. The gradient at each time point was used to calculate the% difference obtained between LDL and HDL.

FIG. 12 shows the difference in plasma LDL (filled circles) and HDL (open circles) using E2C4 (example 9).

FIG. 13 shows the difference in plasma LDL (filled circles) and HDL (open circles) using P2C4 (example 9).

FIG. 14 shows the gradient at each time point used to calculate the% difference between the measurement of LDL and HDL (example 10).

Example 1

LDL buffer #1(Tris buffer-5% glycine pH9.0)

Trizma Pre-Set Crystals, pH9.0 (Sigma), T-1444) was dissolved in 950ml dH₂O(dH₂O ═ deionized water), and the pH was recorded. Subsequently, 50G glycine (Sigma, G-7403) was added to the tris buffer and the pH was recorded. The pH was adjusted to within 8.8-9.2 using 10M potassium hydroxide (Sigma, P-5958) and dH₂The solution was prepared to 1000ml and the final pH (pH9.1) was recorded. The solution was stored at 4 ℃.

Glycol ether solution

A double strength glycol ether solution was prepared using LDL buffer # 1.

Diethylene glycol monopentyl ether (Sigma-Aldrich, 32285)

About 2.5% (0.0218g in 872. mu.l LDL buffer # 1)

Diethylene glycol monobutyl ether (Sigma-Aldrich, 537640)

About 10% (0.0640g in 640. mu.l LDL buffer # 1)

Scipac LDL and HDL samples

LDL (Scipac, P232-8) and HDL (Scipac, P233-8) samples were made 10-fold higher in concentration (due to a 1: 10 dilution in the final test mixture) using delipidated serum (Scipac, S139). The samples were then analyzed using a Space clinical analyzer (Space clinical analyzer) (Schiappanelli Biosystems Inc).

Enzyme mixture

The enzyme mixture was made 2-fold stronger using LDL buffer #1

160mM ruthenium (III) hexamine chloride (Alfa Aesar, 10511)

17.7mM thionicotinamide adenine dinucleotide (Oriental Yeast Co)

8.4mg/ml Pseudomonas redox protein reductase (Biocatalysts)

6.7mg/ml cholesterol esterase (Sorachim/Toyobo, COE-311)

44.4mg/ml cholesterol dehydrogenase, gelatin free (Amano, CHDH-6)

Test protocol

Mu.l of a double strength 1, 2-ethanediol solution were mixed with 9. mu.l of the enzyme mixture. At-30 seconds T, 2 μ l of sample (10-fold concentrated LDL or HDL, or delipidated serum) was mixed with the resulting glycol ether: the enzyme mixture was mixed and 9. mu.l of the resulting solution was placed on the electrode. When T ═ 0 seconds, chronoamperometric tests were initiated. The oxidation current was measured at 0.15mV at 5 time points (10, 32, 63, 90 and 110 seconds), and the reduction current was measured at-0.45 mV at the final time point. Each sample was tested in duplicate.

Analysis of

These data were analyzed along with the LDL, HDL and delipidated serum concentrations obtained by the spatial analyzer. The gradient of HDL and LDL responses at each time point was used to calculate the% difference obtained from measurement of LDL and HDL.

FIGS. 1 and 2 illustrate the results obtained for selectively solubilizing LDL over HDL when diethylene glycol monopentyl ether was used.

FIGS. 3 and 4 illustrate the results obtained for selectively solubilizing LDL over HDL when using diethylene glycol monobutyl ether.

Conclusion

Diethylene glycol monobutyl ether (5%) showed a preferential differentiation to LDL > 35%. Diethylene glycol monopentyl ether (1.25%) also showed preferential differentiation for LDL, but to a lesser extent > 20%.

Example 2: genzyme cholesterol esterase vs. Genzyme lipase solution

RuAcAc＝[Ru^III(acac)₂(py-3-COOH)(py-3-COO)]

A30 mM Ruacac solution was prepared using a buffer containing 0.1M KCl, Tris pH9.0, 5% glycine.

Diethylene glycol butyl ether solution: the 10% glycol ether solution was made using a RuAcac solution.

The enzyme mixture was made using Ruacac solution:

17.7mM thionicotinamide adenine dinucleotide

8.4mg/ml Pseudomonas redox protein reductase

6.7mg/ml cholesterol esterase or 6.7mg/ml lipase

44.4mg/ml cholesterol dehydrogenase, gelatin free

LDL (Scipac, P232-8) and HDL (Scipac, P233-8) samples were prepared using delipidated serum (Scipac, S139). The samples were then analyzed using a spatial clinical analyzer (Schiappanesis biosystems Inc).

Test protocol

Mu.l of a double strength glycol ether solution (or Ruacac solution without glycol ether) was mixed with 9. mu.l of the enzyme mixture. At T-30 seconds, 2 μ l of sample (LDL or HDL, or delipidated serum) was mixed with the resulting glycol ether: the enzyme mixture was mixed and 9. mu.l of the resulting solution was placed on the electrode. This electrode is described in WO 200356319. When T ═ 0 seconds, chronoamperometric tests were initiated. The oxidation current was measured at 0.15mV at 7 time points (0, 28, 56, 84, 112, 140 and 168 seconds), and the reduction current was measured at-0.45 mV at the final time point. Each sample was tested in duplicate.

Results

Wherein E2C4 is diethylene glycol butyl ether.

The gradient at each time point was used to calculate the% difference obtained from the measurement of LDL and HDL and is shown in FIG. 5.

Conclusion

Differentiation of LDL in the enzyme mixture was conferred by cholesterol esterase or lipase in the presence of 5% diethylene glycol butyl ether, although the differentiation to LDL was the highest with cholesterol esterase.

These data indicate that the differentiation induced using lipase is converted from HDL differentiation to LDL differentiation by the addition of diethylene glycol butyl ether. This shows that diethylene glycol butyl ether has a stronger effect on LDL differentiation than cholesterol esterase.

Example 3: determination of optimal concentration of diethylene glycol butyl Ether to selectively solubilize LDL

The objective of this experiment was to titrate diethylene glycol monobutyl ether to determine the optimal concentration for selective solubilization of LDL with minimal interaction with HDL for the purpose of detecting LDL.

Solution:

RuAcAc solution: 30mM RuAcac was prepared using a buffer containing Tris pH9.0, 10% sucrose and 0.1 MKCl.

Glycol ether solutions were made in the Ruacac solution described above as 12%, 10%, 8%, 6%, 4% and 2% diethylene glycol butyl ether.

The enzyme mixture (containing cholesterol esterase) and the LDL and HDL samples were prepared according to the same formulation as in example 2.

The method comprises the following steps:

the test and analysis were performed according to the method described in test 2.

Conclusion

The gradient of response to LDL increases with increasing concentration of diethylene glycol butyl ether. This resulted in the difference to LDL being the highest at 6% diethylene glycol butyl ether.

Example 4: determination of optimal concentration of dipropylene glycol butyl Ether for Selective solubilization of LDL

The objective of this experiment was to vary the concentration of dipropylene glycol monobutyl ether to determine the optimal concentration for selective solubilization of LDL with minimal interaction with HDL for the purpose of detecting LDL.

Solutions of

30mM Ruacac buffer, enzyme solution (containing cholesterol esterase) and HDL or LDL Scipac samples were prepared as described in example 2.

Glycol ether solutions were made by using 3.5%, 3%, 2.5%, 2%, 1.5% and 1% dipropylene glycol butyl ether in the Ruacac solution described above.

The method comprises the following steps:

the experiment was performed as described in example 2. The results are shown in fig. 7.

Conclusion

An increase in the gradient of response to LDL was obtained as the concentration of dipropylene glycol butyl ether was increased. This results in an increase in differentiation to LDL. The highest differences were obtained at 1.5 and 1.75% dipropylene glycol butyl ether.

Example 5: identification of Agents showing increased Selectivity for LDL

The purpose of this experiment was to identify reagents that exhibit selectivity for LDL with minimal interaction with HDL for the purpose of detecting LDL.

Solutions of

Glycol ether solution: each glycol ether solution was made using Tris buffer, pH9.0, 5% glycine. The following amounts resulted in a double strength glycol ether solution. Note that due to minor variations in weighing, the percentages are only approximate:

2-methoxyethanol (Aldrich 185469)

10% (0.0477g in 477. mu.l buffer)

Triethylene glycol methyl ether (Fluka 90450)

10% (100. mu.l + 900. mu.l buffer)

Diethylene glycol propyl ether (Aldrich 537667)

10% (0.0947g in 947. mu.l buffer)

Diethylene glycol butyl ether (Aldrich 537640)

10% (0.0640g in 640. mu.l buffer)

Diethylene glycol pentyl ether (Fluka 32285)

2.5% (0.0218g in 872. mu.l buffer)

1-methoxy-2-propanol (Aldrich 65280)

10% (0.0459g in 459. mu.l buffer)

Dipropylene glycol butyl ether (Aldrich 388130)

2.5% (0.0121g in 484. mu.l buffer)

Tripropylene glycol methyl ether (Aldrich 30,286-4)

10% (0.0463g in 463. mu.l buffer)

Tripropylene glycol butyl ether (Aldrich 48,422-9)

2.5% (0.0176g in 704. mu.l buffer)

Glycerol ethoxylate-co-propoxylate triol (Aldrich 40, 918-9)

5% (0.0534g in 1.068ml buffer)

Neopentyl glycol ethoxylate (Aldrich 410276)

10% (0.0619g in 619. mu.l buffer)

Propylene glycol propyl ether (Sigma-Aldrich 424927)

10% (0.0444g in 444. mu.l buffer)

1-tert-butoxy-2-propanol (Sigma-Aldrich 433845)

10% (0.0470g in 470. mu.l buffer)

Dipropylene glycol propyl ether (Sigma-Aldrich 484210)

10% (0.0458g in 458. mu.l buffer)

Tripropylene glycol propyl ether (Sigma-Aldrich 469904)

10% (0.0435g in 435. mu.l buffer)

Dipropylene glycol tert-butyl ether (Sigma-Aldrich 593346)

10% (0.0417g in 417. mu.l buffer)

2-propoxyethanol (Sigma-Aldrich 82400)

10% (0.0444g in 444. mu.l buffer)

Scipac LDL and HDL samples: LDL and HDL samples were prepared using delipidated serum.

Enzyme mixture

The enzyme mixture was prepared using the above Tris buffer, ph9.0, 5% glycine, containing:

160mM ruthenium hexamine (III) chloride

17.7mM thionicotinamide adenine dinucleotide

8.4mg/ml Pseudomonas redox protein reductase

6.7mg/ml cholesterol esterase

44.4mg/ml cholesterol dehydrogenase, gelatin free

Test protocol

Mu.l of glycol ether solution was mixed with 9. mu.l of the enzyme mixture. At T-30 seconds, 2 μ l of sample (LDL, HDL, or delipidated serum) was mixed with the resulting glycol ether: the enzyme mixture was mixed and 9. mu.l of the resulting solution was placed on the electrode. This electrode is described in WO 200356319. When T ═ 0 seconds, chronoamperometric tests were initiated. The oxidation current was measured at 0.15mV at 5 time points (10, 32, 63, 90 and 110 seconds), and the reduction current was measured at-0.45 mV at the final time point. Each sample was tested in duplicate.

Results

The data was analyzed and the gradient at the first time point was used to calculate the% difference obtained between the measurement of LDL and HDL. The results are shown in fig. 8.

Example 6: KCl titration 500-1500mM

The purpose of this experiment was to investigate the effect of increased ionic strength on LDL and HDL response in the presence of diethylene glycol monobutyl ether.

Solutions of

30mM Ruacac solution: 30mM RuAcac, Tris pH9.0, 5% glycine, 5% diethylene glycol butyl ether

KCl solutions at 3M, 2M, 1.5M and 1M KCl were made in the Ruacac solution described above.

Enzyme mixtures were made in the Ruacac solution described above:

17.7mM thionicotinamide adenine dinucleotide

8.4mg/ml Pseudomonas redox protein reductase

6.7mg/ml cholesterol esterase

44.4mg/ml cholesterol dehydrogenase

Scipac LDL and HDL samples were prepared in delipidated serum.

Test protocol

Mu.l of KCl solution was mixed with 9. mu.l of the enzyme mixture. At-30 seconds T, 2 μ l of the sample was mixed with acetone; the KCl obtained: the enzyme mixture was mixed and 9. mu.l of the resulting solution was placed on the electrode. When T ═ 0 seconds, chronoamperometric tests were initiated. The oxidation current was measured at 0.15mV at 7 time points (0, 32, 64, 96, 128, 160 and 192 seconds), and the reduction current was measured at-0.45 mV at the final time point. Each sample was tested in duplicate.

The data was analyzed and the gradient at each time point was used to calculate the% difference obtained between the measurement of LDL and HDL. The results are shown in fig. 9.

Conclusion

Increasing KCl concentration to very high concentrations (1.5M) reduced differentiation to LDL by increasing the gradient in response to HDL. At 500, 750 and 1M KCl, high differentiation to LDL was obtained.

Example 7: KCl titration 0-500mM

The purpose of this experiment was to investigate the effect of ionic strength on LDL and HDL response in the presence of diethylene glycol butyl ether.

Solutions of

A30 mM Ruacac solution was prepared in a buffer containing Tris pH9.0, 5% glycine, 5% diethylene glycol butyl ether solution.

KCl solutions were made up to concentrations of 1M, 500mM and 100mM KCl in the Ruacac solution described above.

The enzyme mixture was made to double strength using Ruacac solution:

17.7mM thionicotinamide adenine dinucleotide (Oriental Yeast Co)

8.4mg/ml Pseudomonas redox protein reductase (Biocatalysts)

6.7mg/ml Cholesterol esterase (Genzyme)

44.4mg/ml cholesterol dehydrogenase, gelatin free (Amano, CHDH-6)

Scipac LDL and HDL samples were made in delipidated serum from Scipac.

Test protocol

Mu.l of KCl solution or Ruacac solution (blank) was mixed with 9. mu.l of the enzyme mixture. At-30 seconds T, 2 μ l of sample (10x concentrated LDL or HDL, or delipidated serum) was mixed with the resulting KCl: the enzyme mixture was mixed and 9. mu.l of the resulting solution was placed on the electrode. This electrode is described in WO 200356319. When T ═ 0 seconds, chronoamperometric tests were initiated. The oxidation current was measured at 0.15mV at 7 time points (0, 32, 64, 96, 128, 160 and 192 seconds), and the reduction current was measured at-0.45 mV at the final time point. Each sample was tested in duplicate.

Results

The data was analyzed and the gradient at each time point was used to calculate the% difference obtained between the measurement of LDL and HDL. The results are shown in fig. 10.

Conclusion

Increasing the concentration of KCl in the range of 0-500mM results in a higher differential for LDL with increasing KCl concentration due to an increasing gradient in response to LDL.

Example 8: investigation of the Effect of Ionic Strength on Selective solubilization of LDL

The purpose of this experiment was to investigate the effect of ionic strength on the selective solubilization of LDL under conditions of minimal interaction with HDL, by varying the concentration of ruthenium hexaamine chloride mediator for the purpose of detecting LDL.

Solutions of

Glycol ether solutions containing 12% diethylene glycol monobutyl ether were prepared in Tris buffer (pH9.0, 5% glycine).

Scipac LDL and HDL were made to various concentrations using Scipac delipidated serum.

The enzyme mixture was made to double strength using Tris buffer, pH9.0, 5% glycine.

4 separate enzyme mixtures were prepared containing 80, 160, 240 or 480mM ruthenium hexamine chloride:

80, 160, 240 or 480mM ruthenium hexamine chloride

17.7mM thionicotinamide adenine dinucleotide

8.4mg/ml Pseudomonas redox protein reductase

6.7mg/ml cholesterol esterase

44.4mg/ml cholesterol dehydrogenase, gelatin free

Test protocol

Mu.l of a double strength glycol ether solution were mixed with 9. mu.l of the enzyme mixture. At T-30 seconds, 2 μ l of sample (10x concentrated LDL or HDL, or delipidated serum) was mixed with the resulting glycol ether: the enzyme mixture was mixed and 9. mu.l of the resulting solution was placed on an electrode (which is described in WO 200356319). When T ═ 0 seconds, chronoamperometric tests were initiated. The oxidation current was measured at 0.15mV at 5 time points (0, 28, 56, 84 and 112 seconds) and the reduction current was measured at-0.45 mV at the final time point. Each sample was tested in duplicate.

Analysis of

The data was analyzed and the gradient at each time point was used to calculate the% difference obtained between LDL and HDL. The results for the first time point 0 seconds are shown in table fig. 11 a-d.

Conclusion

The highest difference to LDL was obtained with 80mM ruthenium hexamine chloride.

While not wishing to be bound by any particular theory, it is postulated that changes in the level of ions present alter the relative solvating power of the co-solvent for cholesterol until the ionic strength or ionic concentration reaches a solubility-limited level.

Example 9: plasma calibration using diethylene glycol butyl ether or dipropylene glycol butyl ether

The purpose of this experiment was to study the response to plasma LDL and HDL responses in the presence of diethylene glycol monobutyl ether (E2C4) or dipropylene glycol monobutyl ether (P2C 4).

Solutions of

KCl buffer solution: tris buffer pH9.0, 5% glycine, 0.2M KCl

40mM Ruacac was prepared using the KCl buffer described above.

A3M KCl solution was prepared in the above Ru acac solution.

Enzyme mixture: the enzyme mixture (without co-solvent) was made using Ruacac solution:

17.7mM thionicotinamide adenine dinucleotide

8.4mg/ml Pseudomonas redox protein reductase

6.7mg/ml cholesterol esterase

44.4mg/ml cholesterol dehydrogenase, gelatin free

Enzyme mix containing 12% E2C 4: 0.0304g E2C4(Sigma-Aldrich) was dissolved in 253. mu.l of the enzyme mixture.

Enzyme mix containing 3.5% P2C 4: 0.0075g P2C4(Sigma-Aldrich) was dissolved in 250. mu.l of the enzyme mixture.

Plasma sample: frozen plasma samples were thawed for at least 30 minutes, followed by 5 minute centrifugation. The samples were then analyzed using a spatial clinical analyzer (Schiappandelli Biosystems Inc).

Test protocol

For the enzyme mixture containing E2C4, 1.5. mu.l of 3M KCl solution was mixed with 7.5. mu.l of the enzyme mixture. At-30 seconds, 9 μ l of the sample (plasma, or delipidated serum) was mixed with the resulting KCl: the enzyme mixture was mixed and 9. mu.l of the resulting solution was placed on the electrode. When T ═ 0 seconds, chronoamperometric tests were initiated. The oxidation current was measured at 0.15mV at 7 time points (0, 32, 64, 96, 128, 160 and 192 seconds), and the reduction current was measured at-0.45 mV at the final time point. Each sample was tested in duplicate.

For the enzyme cocktail containing P2C4, 9 μ l of the enzyme cocktail was mixed with 9 μ l of the sample (plasma, or delipidated serum) at T ═ 30 seconds. Mu.l of the resulting solution was placed on an electrode and a chronoamperometric assay was initiated as described above for E2C4 at T0 seconds.

Analysis of

The data is analyzed. The gradient at each time point was used to calculate the% difference obtained between the measurement of LDL and HDL.

Results

With E2C4, the difference to plasma LDL was 103% at time t ═ 0 seconds (fig. 12-HDL indicated by open circles, LDL indicated by filled circles). With P2C4, the difference to plasma LDL was 91% (fig. 13-HDL indicated by open circles, LDL indicated by filled circles) at time t 96 seconds.

Conclusion

High differentiation to plasma LDL was obtained with E2C4 or P2C 4.

Example 10: assay for determining a reagent that selectively solubilizes LDL under conditions that have minimal interaction with HDL for the purpose of detecting LDL

Solutions of

0.1M KCl buffer-Tris buffer, pH9.0, 5% glycine, 0.1M KCl

Glycol ether solution

Glycol ether solutions of double strength were prepared using 0.1M KCl solution:

diethylene glycol butyl ether (Aldrich 537640)

10% (0.0958g in 958. mu.l KCl buffer)

Enzyme mixture:

enzyme mixtures were prepared using 0.1M KCl and contained:

40mM RuAcac

17.7mM thionicotinamide adenine dinucleotide

8.4mg/ml Pseudomonas redox protein reductase

6.7mg/ml cholesterol esterase

44.4mg/ml cholesterol dehydrogenase, gelatin free

Scipac LDL and HDL samples:

LDL (Scipac, P232-8) and HDL (Scipac, P233-8) samples were prepared at 10X concentration required using delipidated plasma (Scipac, S139). The samples were then analyzed using a spatial clinical analyzer (Schiappandelli Biosystems Inc).

Test protocol

Mu.l of glycol ether solution was mixed with 9. mu.l of the enzyme mixture. At T-30 seconds, 2 μ l of sample (LDL or HDL, or delipidated serum) was mixed with the resulting glycol ether: the enzyme mixture was mixed and 9. mu.l of the resulting solution was placed on an electrode (the electrode is described in WO 200356319). When T ═ 0 seconds, chronoamperometric tests were initiated. The oxidation current was measured at 0.15mV at 5 time points (0, 35, 63, 90, 118, 145 and 172 seconds), and the reduction current was measured at-0.45 mV at the final time point. Each sample was tested in duplicate.

Analysis of

These data were analyzed along with the concentrations of LDL, HDL and delipidated serum obtained by the spatial analyzer. The gradient at each time point was used to calculate the% difference obtained between the LDL and HDL measurements. The results are shown in fig. 14.

Conclusion

High differentiation to LDL was obtained with diethylene glycol butyl ether.

Claims (30)

1. A biosensor comprising a substrate containing a biochemical analyte, an enzyme system, a low molecular weight glycol ether and a detection means.

2. A biosensor as claimed in claim 1 wherein the substrate is a biological fluid such as blood, serum or plasma.

3. The biosensor of claim 2, wherein the biochemical analyte determined from the biological fluid is a lipoprotein.

4. The biosensor in accordance with claim 3, wherein said lipoprotein is a low density lipoprotein.

5. A biosensor as claimed in any one of the preceding claims wherein the enzyme system comprises a cholesterol enzyme such as cholesterol esterase, cholesterol oxidase or cholesterol dehydrogenase.

6. A biosensor as claimed in any one of the preceding claims wherein the low molecular glycol ether is selected from the group having 1-4 repeating linear or branched alkylene groups.

7. The biosensor in claim 6, wherein the alkylene group is 1, 2-ethylene, 1, 2-propylene and its isomers, butylene and its isomers, or pentylene and its isomers, or a combination thereof.

8. The biosensor of any of the preceding claims, wherein the glycol ether is substituted with an alkyl group, the alkyl group optionally substituted with one or more alkoxy groups.

9. The biosensor in claim 8, wherein said alkyl group is C₁-C₅An alkyl group.

10. The biosensor as set forth in any one of claims 6 to 9, wherein the alkylene group or the alkyl group is substituted with 1 to 4 alkoxy groups.

11. The biosensor in claim 11, wherein the 1-4 alkoxy groups are 1-4 ethoxy groups.

12. The biosensor in any of the preceding claims, wherein the low molecular weight glycol ether is 2-methoxyethanol, tripropylene glycol methyl ether, diethylene glycol propyl ether, diethylene glycol butyl ether, diethylene glycol pentyl ether, 1-methoxy-2-propanol, dipropylene glycol butyl ether, tripropylene glycol butyl ether, glycerol ethoxylate-co-propoxylate triol, neopentyl glycol ethoxylate, propoxyethanol (propxyethane), triethylene glycol methyl ether, propylene glycol propyl ether, 1-tert-butoxy-2-propanol, dipropylene glycol propyl ether, tripropylene glycol propyl ether or dipropylene glycol tert-butyl ether.

13. The biosensor of any of the preceding claims, wherein the biosensor further comprises an aqueous buffer solution.

14. The biosensor in claim 13, wherein the buffer solution typically has an alkaline pH.

15. The biosensor in any one of claims 1 to 14, wherein the ionic strength of the solution is increased so as to improve selectivity for low density lipoprotein.

16. The biosensor in claim 15, wherein the ionic strength of the solution is increased by adding a salt selected from the group consisting of: potassium chloride, magnesium sulfate, ruthenium hexamine chloride, sodium chloride, calcium chloride, magnesium chloride, lanthanum chloride, sodium sulfate, or magnesium sulfate.

17. A biosensor as claimed in any one of the preceding claims wherein the detection means is in the form of an electrochemical cell.

18. A detection system for measuring the amount of a biochemical analyte in a sample, the detection system comprising the steps of:

a) providing a mixture of a low molecular weight glycol ether solution and an enzyme mixture;

b) adding a solution of a sample to be detected;

c) incubating the resulting mixture under conditions that cause a change in the measurable signal;

d) measuring the resulting change; and

e) the amount of analyte or the difference between HDL and LDL is determined in the raw sample using a calibration curve.

19. The test system as claimed in claim 18, wherein the analyte is low density lipoprotein.

20. The detection system of claim 18 or 19, wherein the measurable signal is an electrochemical signal, a colorimetric signal, a thermal signal, a piezoelectric signal, or a spectroscopic signal.

21. A detection system as claimed in any of claims 18 to 20 wherein the low molecular weight glycol ether is as defined in any of claims 6 to 12.

22. The test system as claimed in any one of claims 18 to 21, wherein the biological analyte and reagents are dried prior to use.

23. Use of a low molecular weight glycol ether for solubilising a biochemical analyte.

24. Use as claimed in claim 23 wherein the low molecular weight glycol ether is as defined in any one of claims 6 to 12.

25. The use as defined in any one of claim 23 or claim 24, wherein the glycol ether is for solubilizing lipoproteins, such as low density lipoprotein cholesterol.

26. Use of a salt to increase the ionic strength of a solution containing low density lipoprotein, high density lipoprotein and glycol ether, wherein an increase in the ionic strength of the solution modulates the relative solubilities of the low density lipoprotein and the high density lipoprotein.

27. The use as claimed in claim 26, wherein the increase in ionic strength increases the solubility of the low density lipoprotein relative to the high density lipoprotein.

28. The use as claimed in either of claim 26 or claim 27 wherein the salt is selected from the group consisting of potassium chloride, magnesium sulfate, ruthenium hexamine chloride, sodium chloride, calcium chloride, magnesium chloride, lanthanum chloride, sodium sulfate or magnesium sulfate.

29. The use as claimed in any one of claims 26 to 28 wherein the concentration of the salt is in the range 0.1M to 1M.

30. The use as claimed in any one of claims 26 to 28 wherein the ionic strength of the solution is in the range 0.5M to 1.5M.