Immobilized carbohydrate biosensor

Details Immobilized carbohydrate biosensor Immobilized carbohydrate biosensor

1994-12-19

2001-05-15

Chin, Christopher L. (Department: 1641)

Chemistry: electrical and wave energy

Apparatus

Electrolytic

C204S403060, C356S445000, C422S068100, C422S082010, C422S082050, C422S082120, C435S005000, C435S007100, C435S007200, C435S007320, C435S007800, C436S501000, C436S164000, C436S169000, C436S805000, C436S806000, C436S827000

Reexamination Certificate

active

06231733

ABSTRACT:

The present invention relates to a biosensor in which a carbohydrate or a derivative thereof is used to generate a detectable signal via the specific binding of a protein, a virus or a cell.
BACKGROUND
Biosensors are characterised by a physical or chemical signal transducer, which response is activated by a specific interaction between a biochemical structure (which directly or indirectly has been bound to the transducer) and one or several analytes.
Biosensors are used to detect the analyte/analytes and in certain cases also for quantification of the analyte/analytes.
The advantages of the biosensor are that a physical or chemical transducer has been made specific so that a general physical or chemical parameter (e.g. temperature, pH, optical density) can be used for the detection of one specific substance in a complex mixture of non-specific substances.
The limitations of the biosensor are the specificity of the biochemical structure bound to the transducer, the range of specificity and stability, and, that the transducer signal has to be made independent of the background changes in the parameter that the transducer is measuring. In Methods of Enzymology, volume 137, several articles are describing different aspects of biosensors.
DEFINITIONS
Biosensor—physical or chemical signal transducer, e.g. photometer, chemical electrode, temperature or pressure signal transducer, which directly or indirectly has been connected with a biochemical structure. In previous biosensors one has preferentially used an enzyme, a specific protein or antibody as the biochemical structure and in this way the biosensors have been given the property of being able to detect substances which specifically bind to the biochemical structure in a qualitative or quantitative way.
Reflection measurement—measurement of the intensity of light reflected from a surface where the properties of the surface influences the reflection, e.g. biomolecules which change the refraction index of the surface.
Polarisation measurement—measurement of the polarisation of polarised light, usually as the angle of polarisation, which is depending on the binding of biomolecules, virus or cells.
Surface plasmon spectroscopy—optical physical measurement technique which utilise the surface plasmon condition of thin metal surfaces, which can be used to measure small changes of refraction index with high sensitivity, e.g. as caused by the presence of biomolecules on the surface.
Ellipsometry—optical physical measurement technique which can be used to measure small changes of refraction index at surfaces with high sensitivity, by measuring changes in elliptisity of polarised light, e.g. as caused by the presence of biomolecules on the surface.
Piezoelectric crystal—crystal which frequency can be influenced by changes of mass or pressure which can be measured electrically, for example the change of mass caused by the presence of biomolecule(s), virus or cell(s) bound to the crystal surface.
Electrochemical electrode—measuring device which generates an electrical signal caused by an electrochemical reaction at the electrode which is related to a chemical parameter, e.g. pH, pO
2
, pCO
2
, the values of which can vary because of the presence of analyte(s) in a sample specific for a compound bound to the measuring device.
Thermistor—electrical resistance device which changes resistance with the temperature; biochemical reactions are characterised by e.g. specific values of heat consumption/formation, which can be registered via the thermistor.
A large amount of the carbohydrate sequences present in glycoproteins or in glycolipids, and usually also smaller fragments of these sequences, have shown biospecific binding to proteins, virus or cells.
The present invention describes a biosensor where this specificity is used for determination of such a component in a sample. The invention is characterised by that the carbohydrate or a derivative thereof is bound to a surface in the biosensor.
As carbohydrate, one can use fragments (oligosaccharides) of the carbohydrate sequences found in glycoproteins or in glycolipids and one can also use smaller fragments of these sequences, i.e. disaccharide, trisaccharide, tetrasaccharide or a pentasaccharide, because this size usually is sufficient for the oligosaccharide to bind a protein, virus or a cell in a biospecific manner. A review or such carbohydrate sequences can be found in e.g. Chemistry and Physics of Lipids, vol. 42, p. 153-172, 1986, and in Ann. Rev. Biochem., vol. 58, p. 309-350.
The oligosaccharide is usually modified in the reducing end with an aglycon, which is composed of a glycosidically bound organic group which is suitable for binding to the surface in the biosensor. Examples of aglycons are OEtSEtCONHNH
2
,—OEtSPhNH
2
, etc. The binding to the surface in the biosensor can be done directly or via a proteins, e.g. bovine serum albumine or via a chemical structure which has been adsorbed or which has been covalently bound to the surface. Such a chemical structure can contain reactive organic groups such as carboxyl-, sulfonate, cyanate, epoxy-, aidehyde groups or other groups suitable for chemical conjugation with for example an amine or thiol group in the aglycon.
More specific examples of analytes which can be analysed with biosensor according to the present invention are lectins, antibodies against carbohydrates, pathogenic virus or bacteria, such as urinary tract bacteria (e.g. P-fimbriated
E. coli
) or pathogens of the respiratory tract, and bacteria which cause infections/diarrhea in in gastrointestinal tract.
Non-limiting examples of carbohydrate structures of interest and which can be used in the form of a carbohydrate derivative in a biosensor according to the invention, are monosaccharides, disaccharides, trisaccharides and higher oligosaccharides which show biological activity or which has the ability to specifically bind one or more biomolecules or a group of biomolecules. Examples of biomolecules are other saccharides, peptides and proteins. Examples of such carbohydrate sequences are the blood group determinants (for example A, B, H, Lewis-a, Lewis-b, Lewis-x, Lewis-y), cancer-associated carbohydrate sequences, carbohydrate sequences (often di-, tri- or tetrasaccharides) which bind to pathogenic bacteria/toxins or virus of for example the respiratory, the gastrointestinal or the urinary tract, carbohydrate sequences which bind to proteins/cells/white blood cells associated with inflammatory reactions (for example selectin-carbohydrate reactions).
These and other carbohydrate structures which can be used in a biosensor according to the present invention often contain one or more of the following monosaccharides (or a derivative or an analog of any of these) which are &agr;- or &bgr;-glycosidically bound: hexosamine, fucose, mannose, glucose, N-acetyl-glucosamine, N-acetyl-galactosamine, xylose, galactose, or another monosaccharide. These components are usually present in for example pyranose or furanose form.
Examples of carbohydrate derivatives are derivatives where the carbohydrate or a derivative or an analog, are modified in the reducing end with an O-, N-, C- or S-glycosidically bound aglycon which can be an aliphatic or an aromatic compound, an amino acid-, peptide- or protein molecule or a derivative thereof. The aglycon part can thus be composed of for example an O-, N-, C- or S-glycosidically bound aliphatic or aromatic compound which is bound to an amino acid-, peptide- or protein molecule or a derivative thereof. Examples of carbohydrate derivatives which can be used according to the invention are structures in which one or more of the hydroxyl groups in the carbohydrate, in addition to or instead of the hydroxyl group in the reducing end of the carbohydrate part, have been modified with an organic or inorganic group. This can be of interest, for example to increase/modify the biological activity or to facilitate the binding to the biosensor surface according to the invention.
The aglycon part or another group can be used for adsorption or covalent binding of t

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