WO2003072665A1

WO2003072665A1 - Coating for biosensors

Info

Publication number: WO2003072665A1
Application number: PCT/NL2003/000147
Authority: WO
Inventors: Cornelis Gerardus Josephus Koopal
Original assignee: Nederlandse Organisatie Voor Toegepast-Natuurwetenschappelijk Onderzoek Tno
Priority date: 2002-02-28
Filing date: 2003-02-26
Publication date: 2003-09-04
Also published as: NL1020090C2; AU2003215963A1

Abstract

The invention relates to a coating for coating a hydrophobic surface with a polysaccharide hydrogel, wherein a crystalline protein layer is applied between the hydrophobic surface and the hydrogel. The polysaccharide hydrogel is preferably dextran and the crystalline protein layer preferably originates from surface proteins of bacteria, e.g., Lactobacillus. The coating is used in biosensors for measurements in not completely clean solutions, e.g. milk.

Description

COATING FOR BIOCENSORS

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

To measure concentrations of specific biological molecules in a solution that has possibly been obtained directly from an organism, in general sensitive assays are developed that can specifically recognize a particular molecule. In this type of measurements, use is often made of so- called biosensors that convert the biological signal to another signal (such as for example an electric, optical or other type of signal) making the presence of the biological molecule qualitatively or, preferably, quantitatively measurable.

A specific type of biosensors are those sensors wherein the detection of the biological molecule is carried out 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. For this purpose, the biosensors have been provided with a layer of a metal, preferably an inert noble metal such as gold, optionally covered by a glass layer, on which a specific coating has been applied, to which, in turn, coating receptor molecules are adsorbed such as antibodies or enzymes that can 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. US 5,436,161, for example, describes a coating comprising a hydrogel consisting of dextran or cross-linked dextrans, which, after activation, is suitable for binding specific detection molecules such as antibodies or other proteins. It has been found, however, that with this type of coatings (consisting of a weak cation exchanger), in not completely clean solutions (for example a diluted milk solution), non-specific signals are measured, which often partly or wholly undo the effect of the measuring signal. Another disadvantage of this method is that the polysaccharide layer cannot be applied directly to the metal surface, but that first monolayer of an organic molecule (for example a disulfide, diselenide or thiol-containing compound) needs to be applied to the surface to serve as anchorage 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, wherein a crystalline protein layer is applied between the hydrophobic surface and the hydrogel removes the disadvantages of the known coatings.

Crystalline proteins and the possibility to apply these as a layer are already known per se from the literature. The patent US 5,028,335 shows that the 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 be applied as a crystalline network in a layer on a surface. The patent US 6,296,700 demonstrates that this S-layer can be applied as a monolayer to level, hydrophobic surfaces by means of recrystallization. The side that was originally located on the outside of the bacteria now faces the hydrophobic surface and the (more hydrophilic) side that originally constituted the inside of the bacterial surface layer is turned outwards. Consequently, the S-layer can serve as a basis to immobilize specific detection molecules and would thus be suitable as a coating for biosensors.

Surprisingly, it has now been found that an S-layer applied to a hydrophobic surface of the biosensor can bind a polysaccharide hydrogel, thereby strongly reducing the aspecific interaction of the biosensor in not completely clean solutions. This makes it possible to apply the favorable properties of a biosensor with a polysaccharide hydrogel in solutions of, for example, milk.

The S-layer is preferably obtained from bacteria of the Lactobacillus plantarum genus. It has been found that the surface proteins of this bacterium crystallize well on a hydrophobic surface, thereby forming 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 being reduced.

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

Polysaccharides such as agarose, carrageenan, cellulose, starch or derivatives thereof such as polyvinyl alcohol, polyacrylic acid, polyacrylamide and polyethylene glycol can be used as polysaccharide hydrogel. Preferably, however, dextran (for example Dextran T500,

Pharmacia) is used since it already finds wide application in the use as a matrix for binding biomolecules in chromatography and thus much is known about the properties of dextran and the binding of specific receptors thereto. Furthermore, the polysaccharides can be modified so that they 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 means of, for example, a treatment with bromine acetic acid. Other methods of providing carboxyl or other groups are sufficiently well known to a skilled person. Carboxymethyl dextran that already contains carboxyl groups (e.g.

CM-Dextran, Pharmacia) can also be used as a basis.

The dextran layer is covalently bound to the S-layer. For this purpose, the carboxyl groups of the dextran are first activated by, for example, treatment with, for example, EDC (N- (3, dimethyl amino propyl)- N'-ethylcarbodiimide hydrochloride) in a 10 mM MES buffer of pH=6. In a 50 mM borate buffer at pH 8.5, the resultant carboxymethyl dextran is contacted with the outward hydrophilic side of the S-layer crystallized on the metal, thereby covalently coupling the dextran to the S-layer.

The coating according to the invention is specifically applicable in biosensors for use in not completely clean solutions, such as for example a (diluted) milk solution. Besides milk, other not completely clean solutions, which may or may not be diluted, in which the biosensor according to the invention is usable can also be mentioned, such as body fluids such as blood, urine, lymph fluid, cerebrospinal fluid and the like, and foods such as yogurts, cottage cheese, fruit juices, soups, soft drinks, energy drinks, vegetable cooking water, broths, beer and the like.

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

Measuring concentrations of progesterone in cow's milk is important to detect the moment of ovulation ('estrus'). This is highly important in modern dairy farming, where control of livestock reproduction needs to be as efficient as possible. Improving the detection of the ovulation moment will result in more efficient dairy farming through the increase of the duration of the milk yield per cow, the reduction of breeding costs due to failed insemination attempts and the reduction of the number of animals slaughtered due to decreased productivity. The progesterone content in raw milk is a good parameter to demonstrate the ovulation moment in a cow. This progesterone content should be measured during milking, whereby a biosensor according to the invention is an improvement over the current methods.

As a specific recognition molecule for progesterone, specific antibodies are used. These are sufficiently well known and can be readily obtained by a skilled person. 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 are specifically responsible for recognition, for example the N chains, Fab- fragments or the Complementary Determining Regions (CDRs) can be used. As an alternative, progesterone receptors such as they occur 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 standard pattern and, in addition to a ligand-binding portion, also contain a DΝA-binding domain. In principle, the use of the progesterone receptor in the biosensor according to the invention only requires the ligand-binding domain.

Hereinbelow, examples will be given of a coating according to the invention and the use of such a coating in a biosensor. The invention is not limited to the specifically mentioned examples, however, and other embodiments will be clear to a skilled person after reading the description.

Examples

Example 1 - Preparation of S-protein Source

S-layer protein can be produced by L. acidophilus ATCC 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.

Growing the ATCC 4356 strain

A sample of L. acidophilus ATCC 4356 (bead, 70 K) in 5 ml of MRS medium was inoculated under anaerobic conditions at 37°C for 17-24 minutes (incubation oven, not stirred or shaken). The inoculum was transferred to 500 ml MRS (dilution 1 : 100) and grown further under the same conditions. The cells were harvested as soon as the optical density of the medium had reached 0.7; OD695 was measured in relation to a clean MRS medium.

Harvesting L. acidophilus ATCC 4356 and concentrating the S-layer protein

The cultured cells were centrifuged (9000 rpm, 20 minutes at 4 °C), the supernatant was poured and the pellet was washed with 100 ml cold milliQ. The washed pellet was resuspended in 50 ml 1 M LiCl, incubated for 30 hours at room temperature and centrifuged again (18000 rpm, 15 min.,

4 °C). The supernatant was isolated and the pellet was resuspended in 25 ml 5 M LiCl, followed by incubation for 60 minutes at room temperature, and finally the suspension was centrifuged again (18000 rpm, 15 min., 4 °C). Collected supernatant was filtered over 0.2 μm and directly used in the next step (see example 2) or stored cold (4 °C)

Example 2 - Applying an S-layer to a metal surface

A glass plate having thereon a gold layer of approximately 50 nm was cleaned by means of, in succession, a freshly prepared mixture of concentrated sulphuric acid and 30 wt.% hydrogen peroxide (3:1) and a microwave-supported plasma etcher (oxygen argon plasma; 10 vol.% oxygen). After the etch treatment, the glass plate with the gold layer was held in the vapor of boiling ethanol for 1 minute and then dried under a nitrogen gas flow.

S-layer protein was isolated from a stock solution in 5 M LiCl by means of ultrafiltration over a 10 kD ultrafilter (Omegafilter, Filtron). The isolation was carried out by concentrating 10 ml of the LiCl to 1 ml and then introducing the residue in lOmM Tris buffer pH 7.5. Through re- concentration to 1 ml and re-introduction in 10 ml Tris buffer, the isolated S-layer solution was rinsed. This was repeated 4 times to lose all LiCl. The concentration of S-protein could then be determined spectrophotometrically and, if required, the solution was diluted to an S-protein concentration of 100 μg/ml. The isolated S-protein solution (100 μg/ml) was applied to the gold surface in a quantity of approximately 100 μl per cm². Then, the surface with the S-protein thereon was incubated for a minimum of three hours at room temperature in an atmosphere of high humidity. Upon expiry of the incubation time, the surface was rinsed with milliQ-water and dried under a nitrogen gas flow.

Example 3 - Applying dextran layer to S-layer

Carboxymethyl dextran 1.5 g (CM-Dextran, sodium salt, Fluka 27560) was dissolved in 10 ml MES buffer 10 mM, pH 6, having dissolved therein 400 mM EDC and 100 mM NHS (N-hydroxysuccinimide). After the dextran had been dissolved completely, the solution was stirred for 30 minutes at room temperature. Then, the obtained solution was applied to the gold layer modified with the S-protein in a quantity of approximately 100 μl per cm². Then, the surface with the CM-dextran thereon was incubated for a minimum of three hours at room temperature in an atmosphere of high humidity. After the incubation time had passed, the surface was rinsed with milliQ-water and dried under a nitrogen gas flow.

Example 4 - Applying anti-SEB to a modified sensor surface

The gold-S-protein-CM-dextran sensor surface was incubated for 30 minutes at room temperature 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 μg/ml anti-SEB (SEB: Staphilococcus Enterotoxin B) in a buffer of lOmM sodium acetate, pH 4.8 was applied to the modified sensor surface and again incubated for 30 minutes. After the coupling of the antibody to the sensor surface had been completed, the solution was removed from the sensor surface and a solution of 1 M ethanolamine in water was applied to the surface for 1 minute to deactivate the excess of activated carboxyl groups and then the modified sensor surface was rinsed with milliQ water and finally dried under a nitrogen gas flow.

Example 5 — Comparative measurements of new sensor surface in relation to Biacore CM5 chip

A gold-S-protein-CM-dextran-anti-SEB sensor surface was applied in a Biacore 1000 (Biacore, Sweden) and rinsed with HBS buffer (hepes- buffered saline, pH 7.2) until a stable baseline was established. SEB concentrations in HBS buffer in a range of 20-2000 ng/ml were presented to the modified sensor surface and the response was registered. After each measurement, the antibodies occupied by SEB were cleared for the next measurement by rinsing the sensor surface with a glycine-HCl buffer (50 mM, pH 1.85). The observed response at each separate SEB concentration was plotted as a function of the logarithm of the SEB concentration involved. The result is shown in the figure below.

For comparison, the figure also shows a calibration curve of identical measurements carried out with a commercially available CM5 chip (Biacore, Sweden). In accordance with example 3, the CM5 chip was provided with anti-SEB bodies in a similar manner. The figure shows that the sensitivities for measuring low concentrations of SEB are highly similar in both systems, although the sensor according to the present invention seems slightly more sensitive to lower concentrations of SEB, and thus has a lower detection limit.

Claims

1. A 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. A coating according to claim 1, characterized in that the crystalline protein layer consists of bacterial surface proteins.

3. A coating according to claim 2, characterized in that the surface proteins are originate from Lactobacillus plantarum.

4. A coating according to any one of the preceding claims, characterized in that the polysaccharide hydrogel comprises dextran.

5. A coating according to any 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 is applicable in a biosensor.

7. A biosensor comprising a coating according to any one of claims 1-5.

8. A biosensor according to claim 7, characterized in that the biosensor is suitable for measurements in not completely clean liquids.

9. A biosensor according to any one of claims 7 or 8, characterized in that the biosensor is suitable for measurements in milk.

10. A biosensor according to any one of claims 7-9, characterized in that the biosensor is suitable for measuring progesterone contents.

11. A method for measuring concentrations of substances in milk, characterized in that a biosensor according to any one of claims 7-9 is used.

12. A method according to claim 11, characterized in that the substance is progesterone.