NL1020090C2 - New coating for biosensors. - Google Patents

Description

VO

Title: New coating for biosensors

The invention is in the field of coatings for biosensors, in particular for the use of biosensors in not completely clean solutions.

For measuring concentrations of specific biological molecules in a solution that is possibly directly obtained from an organism, sensitive assays are generally developed that can specifically recognize a particular molecule. In this type of measurement use is often made of so-called biosensors that convert the biological signal into another signal (such as, for example, an electrical, optical or other type of signal), whereby detection of the presence of the biological molecule becomes qualitatively or, more preferably, quantitatively measurable.

A specific type of biosensors are those sensors in which the detection of the biological molecule is done by means of 'surface plasmon resonance' (SPR) which is based on detection of changes in the optical properties of a surface layer as a result of the interaction of a 'receptor' with the medium to be measured.

To this end, the biosensors are provided with a layer of a metal, preferably an inert noble metal such as gold, whether or not covered with a glass layer, on which a specific coating is applied, to which in turn receptor molecules are attached such as antibodies or enzymes that are specifically recognize and bind a biological substance.

A coating for coating a hydrophobic surface such as glass or metal comprising a polysaccharide hydrogel is already known from the literature. For example, US 5,436,161 describes a coating comprising a hydrogel consisting of dextran or cross-linked dextrans, which after activation is suitable for binding the specific detection molecules such as antibodies or other proteins. However, it appears that with this type of coatings 1020090 2 (consisting of a weak cation exchanger) non-specific signals are measured in solutions that are not completely clean (for example a diluted milk solution), which often partially or completely cancel out the effect of the measurement signal. Another disadvantage of this method is that the polysaccharide layer cannot be applied directly to the metal surface, but first a single layer of an organic molecule (for example a disulfide, diselenide or thiol-containing compound) must be applied to the surface in order to to serve as an anchor for the polysaccharide hydrogel.

The present invention provides a solution to these problems.

It was found that a coating for coating a hydrophobic surface comprising a polysaccharide hydrogel, in which a crystalline protein layer is applied between the hydrophobic surface and the hydrogel, eliminates the disadvantages of the known coatings.

Crystalline proteins and the possibility of applying them as a layer are already known per se from the literature. The patent US 5,028,335 shows that the surface layer (surface layer or S-layer) of cell envelopes of some prokaryotes or fungi, such as for example the Bacilleae, consisting of proteins or protein-containing molecules, can be separated from the underlying cell layers and as a crystalline network can be applied in a layer on a surface. U.S. Pat. No. 6,296,700 shows that this S layer can be applied as a "monolayer" on flat hydrophobic surfaces by recrystallization. Here, the side that was originally in the bacteria on the outside has now turned to the hydrophobic surface and the (more hydrophilic) side that originally formed the inside of the bacterial surface layer has turned outwards. As a result, this S-layer can serve as a basis for immobilizing specific detection molecules and would therefore be suitable as a coating for biosensors.

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It has now surprisingly been found that an S layer applied to the hydrophobic surface of the biosensor can bind a polysaccharide hydrogel so as to greatly reduce the non-specific interaction of the biosensor in not completely clean solutions. This makes it possible to use the favorable properties of a biosensor with a polysaccharide hydrogel in solutions of, for example, milk.

Preferably, the S layer is obtained from bacteria of the genus Lactobacillus plantarum. It has been found that the surface proteins of this bacterium crystallize well on a hydrophobic surface and thereby form a single continuous layer. It has also been found that this layer can be modified (for binding with the polysaccharide hydrogel) without the crystallizing properties diminishing.

Prior to applying the S layer, the metal surface is preferably first treated with ethanol, thereby providing a clean and fat-free surface layer to which the protein layer can adhere well.

As polysaccharide hydrogel, polysaccharides such as agarose, carrageenan, cellulose, starch or derivatives thereof such as polyvinyl alcohol, polyacrylic acid, polyacrylamide and polyethylene glycol can be used. Preferably, however, dextran (for example Dextran T500, Pharmacia) is used since it is already widely used in the use as a matrix for the binding of biomolecules in chromatography and so much is already known about the properties of dextran and the binding of specific receptors to that. Furthermore, the polysaccharides can be modified to contain groups to which receptors can easily be immobilized, such as hydroxyl, carboxyl, amino, aldehyde, carbonyl, epoxy or vinyl groups. In the case of dextran, carboxyl groups can be introduced by, for example, treatment with bromoacetic acid. Other methods of introducing carboxyl or other groups are well known to those skilled in the art.

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It is also possible to use carboxymethyldextran (e.g. CM-Dextran, Pharmacia) which already contains carboxyl groups.

The dextran layer is covalently bonded to the S layer. To this end, the carboxyl groups of the dextran are first activated by, for example, treatment with, for example, EDC (N- (3, dimethylamino-propyl) -N'-ethylcarbodiimide hydrochloride) in a 10 M MES buffer of pH = 6. The resulting product carboxymethyl dextran is contacted in a 50mM borate buffer at pH 8.5 with the outward facing hydrophilic side of the metal-crystallized S-layer thereby covalently coupling the dextran to the S-layer.

Specifically, the coating according to the invention can be used in biosensors for use in not completely clean solutions, such as, for example, a (diluted) milk solution. In addition to milk, other not completely clean solutions in diluted or non-diluted form can be mentioned in which the biosensor according to the invention can be used, such as body fluids such as blood, urine, lymph fluid, cerebrospinal fluid, and the like and foods such as yoghurts, quark, fruit juices , soups, soft drinks, energy drinks, cooking fluid from vegetables, broths, beer and the like.

It is of course entirely dependent on the specific recognition molecules (receptors) to be used which biomolecules will be recognized and bound and as such are capable of generating a detectable signal change in the biosensor. As an example, detection of progesterone in cow's milk can be mentioned here, but it is clear to the person skilled in the art that with the aid of the biosensor according to the invention all biomolecules can in principle be detected (provided that specific receptors can be used for this). Examples of such biomolecules are sugars, hormones, antibodies, antigens, antibiotics, (myco) toxins, pesticides, but also environmentally harmful substances such as phosphate-containing and / or nitrate-containing compounds. The invention is particularly well applicable for testing food and / or foodstuffs for the occurrence therein of undesirable substances such as toxins (aflatoxin, fumonisin, atrazine, Staphylococcus aureus enterotoxin B) and hormones (progesterone, clenbuterol, hCG, zearalenone) .

Measuring progesterone concentrations in cow's milk is important to be able to detect the moment of ovulation ('heatiness'). This is of great importance in modern livestock farming where the control of the reproduction of the dairy herd must be as efficient as possible. Improving the detection of the ovulation moment will lead to a more efficient dairy farm by increasing the duration of milk production per cow, reducing breeding costs as a result of unsuccessful insemination attempts and reducing animals killed for non-productivity. The progesterone content in raw milk is a good parameter for demonstrating the ovulation moment in a cow. This progesterone content must be measured during milking and a biosensor according to the invention is hereby an improvement over the existing methods.

Specific antibodies are used as a specific recognition molecule for progesterone. These are sufficiently known and easily available to a person skilled in the art. Antibodies can be used in any desired form: as IgA or IgM polymers, as monomeric portions of these polymers, as IgE or IgG antibodies or as single chain antibodies, but also the fragments of these antibodies that provide specific recognition, e.g. the VL chains, Fab fragments or the Complementary Determinating Regions (CDRs) can be used.

Alternatively, progesterone receptors such as those found in human or animal cells can be used. These receptors are a subclass of the so-called steroid receptors, proteins that are structured according to a fixed pattern and also contain a DNA-binding domain in addition to the ligand-binding part. For the use of the progesterone 1020090 6 receptor in the biosensor according to the invention, in principle only the ligand binding domain is required.

Examples of a coating according to the invention and the use of such a coating in a biosensor are given below. However, the invention is not limited to the specific examples mentioned and other embodiments will be apparent to a person skilled in the art after reading the description.

Examples 10

Example 1 - Preparation of S protein

Source S-layer protein can be produced by L. acidophilus ATCC 15 4356. The protein produced by this microorganism has a molecular weight of 44 kD, has a surplus of positive charge of 15 (+15) and has a pi of 9.40.

Cultivation of the ATCC 4356 strain

A sample of L. acidophilus ATCC 4356 (bead, 70 K) in 5 ml MRS

medium was inoculated under anaerobic conditions at 37 ° C for 17 - 24 hours (incubator, not stirred or shaken).

The inoculum was transferred to 500 ml MRS (dilution of 1: 100) and further cultured under the same conditions. The cells were harvested once the optical density of the medium had reached 0.7; OD695 was measured against clean MRS medium.

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Harvest L. acidophilus ATCC 4356 and concentrate the S-layer protein

The cultured cells were centrifuged (9000 rpm, 20 minutes at 4 ° C), the supernatant was discarded and the pellet was washed with 100 ml of cold milliQ. The washed pellet was resuspended in 50 ml of 1 M LiCl, incubated for 30 minutes at room temperature and recentrifuged (18000 rpm, 15 minutes, 4 ° C). The supernatant was isolated and the pellet was resuspended in 25 ml of 5 M LiCl, then incubated for 60 minutes at room temperature and finally the suspension was centrifuged again (18000 rpm, 15 minutes, 4 ° C). Collected supernatant was filtered over 0.2 µm and used directly in the next step (see Example 2) or stored cold (4 ° C).

Example 2 - applying an S layer on a metal surface 15

A glass plate with a gold layer of about 50 nm on top was cleaned in succession with a freshly prepared mixture of concentrated sulfuric acid and 30% by weight of hydrogen peroxide (3: 1) and a microwave-supported plasma etcher (oxygen margin of plasma; 10% by volume of oxygen). The glass plate with the gold layer was kept in the vapor of boiling ethanol for 1 minute after the etching treatment and then dried with a stream of nitrogen gas.

S-layer protein was isolated from a stock solution in 5 M LiCl by ultrafiltration over a 10 kD ultrafilter (Omega filter, Filtron). The isolation was performed by concentrating 10 ml of the LiCl to 1 ml and then incorporating the residue in 10 mM Tris buffer pH 7.5. The isolated S-layer solution was rinsed by refocusing to 1 ml and taking up again in 10 ml Tris buffer. This was repeated 4 times to get rid of all LiCl. The concentration of S protein could then be determined spectrophotometrically and, if necessary, the solution was diluted to an S protein concentration of 100 pg / ml.

The isolated S-protein solution (100 pg / ml) was applied to the gold surface in an amount of approximately 100 μΐ per cm 2.

Subsequently, the surface with the S protein on it was incubated at RT for a minimum of 3 hours in an atmosphere of high humidity. After the incubation time had elapsed, the surface was rinsed with milliQ water and dried under a stream of nitrogen.

Example 3 - applying dextran layer to S layer

Carboxymethyldextran 1.5 g (CM-Dextran, sodium salt, Fluka 27560) was dissolved in 10 ml of MES buffer 10 mM, pH 6, with 400 mM EDC and 100 mM NHS (N-hydroxysuccinimide) dissolved therein. After the dextran was completely dissolved, stirring was continued for 30 minutes at RT.

The resulting solution was then applied to the gold surface modified with S protein in an amount of approximately 100 μΐ per cm2. Subsequently, the surface with the CM dextran on it was incubated for a minimum of 3 hours at RT in an atmosphere of high humidity. After the incubation time had elapsed, the surface was rinsed with milliQ water and dried under a stream of nitrogen.

Example 4 - Application of anti-SEB on modified sensor surface. The gold S protein CM dextran sensor surface was incubated for 30 minutes at RT with a solution of 400 mM EDC and 100 mM NHS in 10 mM MES pH 6. After removal of the MES buffer, a solution of 50 pg / ml anti-SEB (SEB: Staphilococcus Enterotoxin B) was placed in a buffer of 10 mM sodium acetate pH 4.8 on the modified sensor surface and incubated for another 30 minutes. After the antibody coupling to the sensor surface was completed, the solution was removed from the sensor surface and an aqueous 1 M ethanolamine solution was applied to the surface for 1 minute to deactivate the excess of activated carboxyl groups and then the modified sensor surface rinsed with MilliQ water and finally dried under a stream of nitrogen gas.

Example 4 - Comparison measurements of new sensor surface with respect to 10 Biacore CM5 chip

A gold S protein CM dextran anti-SEB sensor surface was placed in a Biacore 1000 (Biacore, Sweden) and rinsed with HBS buffer (hepes-buffered saline, pH 7.2) until a stable baseline was achieved.

15 SEB concentrations in HBS buffer in a range of 20 - 2000 ng / ml were presented to the modified sensor surface and the response was recorded. After each measurement, the SEB-loaded antibodies were released for the next measurement by flushing the sensor surface with a glycine HCl buffer (50 mM, pH 1.85). The observed response at each individual SEB concentration was plotted as a function of the logarithm of the relevant SEB concentration. The result is shown in Figure 1.

For comparison, the figure also shows a calibration curve of identical measurements performed with a commercially available CM5 chip (Biacore, Sweden). The CM5 chip was similarly provided with anti-SEB antibodies in accordance with Example 3. It can be seen from the figure that the sensitivities for measuring low concentrations of SEB for both systems are very similar, although the sensor according to the present invention appears to be slightly more sensitive to lower concentrations of SEB and this sensor consequently also has a lower detection limit.

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Claims (12)

Coating for coating a hydrophobic surface comprising a polysaccharide hydrogel, characterized in that a crystalline protein layer is applied between the hydrophobic surface and the hydrogel. 2. Coating according to claim 1, characterized in that the crystalline protein layer consists of bacterial surface proteins. 3. Coating according to claim 2, characterized in that the surface proteins are derived from Lactobacillus plantarum, Coating according to one of the preceding claims, characterized in that the polysaccharide comprises hydrogel dextran. Coating according to one of the preceding claims, characterized in that specific detection molecules are cross-linked to the polysaccharide hydrogel. 6. A coating according to any one of the preceding claims, characterized in that the coating can be used in a biosensor. A biosensor comprising a coating according to any one of claims 1-5. Biosensor according to claim 7, characterized in that it is suitable for measurements in not completely clean liquids. Biosensor according to one of claims 7 or 8, characterized in that the biosensor is suitable for measurements in milk. A biosensor according to any one of claims 7-9, characterized in that the biosensor is suitable for measuring progesterone levels. Method for measuring concentrations of substances in milk, characterized in that a biosensor according to any of claims 7-9 is used. Method according to claim 11, characterized in that the substance is progesterone. 1020090