TECHNICAL FIELD

The present invention relates to a stress sensor for detecting a target substance in a fluid comprising one or more sensor units e.g. shaped as cantilevers and comprising a capture coating and a detecting system.

The invention also relates to a method of generating a capture coating for a target substance onto a sensor unit.

BACKGROUND ART

Sensors comprising a sensor unit e.g. in the form of a cantilever and with means for detecting a change of stress in the surface of the cantilever are well known in the art.

In the art of detecting components in fluids, cantilever based sensors with integrated piezo-resistors are used as very . sensitive mechanical stress sensors. As described in e.g. US 6575020 and WO 9938007, micro cantilevers can be used for detection of molecular interaction. At least one surface of the cantilever is coated with a capture layer, which capture layer reacts with a target molecule of interest. If the cantilever is exposed to a sample in which the target molecule is present, the target molecule will react with the capture molecule on the cantilever surface and a surface stress change will be generated.

Due to the surface stress change of the cantilever, a mechanical compression, stretch or decompression is applied to the cantilever and thereby also to the piezo-resistor, and thereby the resistivity of the piezo- resistor changes its value. The mechanical compression or decompression may result in a deflection and/or a stretch and/or a contraction. By measuring the change in resistance, it can be determined whether the target molecule is present in the sample or not, and if so it may also be possible to detect the concentration of the target molecule. Cantilever-based sensors with integrated piezoresistive read-out are described by Thaysen, Ph.D. Thesis, "Cantilever for Bio-Chemical Sensing Integrated in a Microliquid Handling System", September 2001 , Microelektronik Centeret, Technical University of Denmark. Hereby the stress changes on the cantilever sensors can be measured directly by the piezo- resistor. Moreover, integrated read-out greatly facilitates operation in solutions since the refractive indices of the fluids do not influence the detection as it will using optical read-out. Each sensor may have a built-in reference cantilever, which makes it possible to subtract background drift directly in the measurement. Furthermore, by functionalizing the reference cantilever with a "dummy" molecule, non-specific binding events occurring on both the measurement and reference cantilever will be cancelled out in the differential measurement.

The two cantilevers may be connected in a Wheatstone bridge, and the stress change on the measurement cantilever is detected as the output voltage from the Wheatstone, e.g. as described in US 6575020.

It is known to use cantilevers for detecting components in fluids such as gas and liquids. In most situations, the sensors with cantilevers have optical read¬ out, but also sensors comprising cantilevers with integrated piezo-resistors have been described to be useful in detecting components in fluids.

The capture layer of the prior art stress sensors used for detection of a target molecule of interest, normally comprises a chemically functionalized capture surface, which binds to the target substance, whereby a stress is generated.

During recent years a large effort has been put on improving the detection system, by selecting the capture molecules of the capture layer with highest possible selectivity. In PCTDK0300918 is e.g. disclosed a sensor where the target substance and the capture molecule are a specific binding pair.

However, even though the sensor comprises sensor unit and reference unit for subtracting false signal, there is still undesired noise due to bonding of non-target substances to the capture surface. Different groups have tried to improve selectivity by different methods: One example has been to use an array of 8 cantilevers and then coat each of the cantilevers with different polymers. M. K. Bailer, H.P. Lang, J. Fritz, Ch. Gerber, J. K. Gimzewski, U. Drechsler, H. Rothuizen, M. Despont, P. Vettiger, F.M. Battiston, J. P. Ramseyer, P. Fornaro, E. Meyer, H.-J. Gϋntherodt, "A Cantilever Array Based Artificial Nose," Ultramicroscopy,82, 1-9 (2000) disclose a system where the cantilevers have been exposed to different alcohols, solvents etc. and it has been possible to distinguish one component from another. This was done by using the Principal Component Analysis Procedure. This kind of analysis or neural network analysis has proven to be useful if one has to distinguish between two or maximum three different analytes in a sample. But due to the low selectivity using these kinds of polymers, it has been very difficult to distinguish analytes in a multi-analyte environment. Furthermore, if the sensor experiences an unforeseen analyte, the pattern of which is close to one of the analytes of interest, the sensor will return with a false positive signal.

The objective of the invention is thus to provide a stress sensor with an improved selectivity compared with prior art stress sensors.

Another objective is to provide a stress sensor which is relatively simple and cheap to produce, and which can be produced with high uniformity.

These and other objectives, as will be clear from the following description, have been achieved by the invention as defined in the claims.

DISCLOSURE OF INVENTION

The inventor of the present invention has thus discovered that a technology called Molecular Imprinted Polymerizate (MIP) can be used for capturing the target substance on a stress sensor.

The MIP technology is e.g. disclosed in US 5630978 and US 5959050, the contents of which are hereby incorporated by reference. In the MIP technology, a template substance (which may preferably be identical with the target substance) is left to self-assemble with chosen functional monomers and cross linker. Upon polymerization, a cross-linked matrix is formed. Due to the inherent nature of the interactions between the template and the monomers (hydrogen bonding, electrostatic interaction, Van Der Walls forces), the template can afterwards be removed from the matrix leaving functional molecular cavities complementary to the template.

In one embodiment, MIP kind of detector layers can also be obtained by other methods than polymers. As long as it is possible to obtain a stable matrix around the template, which has a relatively high specificity towards the template, it can be used as a MIP type of detector layer. An example is self assembled mono-layers, which also can be used as the matrix.

MIPs are more stable and robust than for example antibodies and they can be stored at room temperature. They can withstand treatment with strong acid or base and can be reused a great number of times without any significant loss in affinity and selectivity.

US2004/0080319 discloses a cantilever with MIP detector layer for detection of molecules due to their mass. The patent describes how the cantilever is attached to a piezoelectric transducer for driving the cantilever in vibration mode. By monitoring the change in resonant frequency, it is possible to calculate the mass attached to the cantilever.

Thanks to the inventor of the present invention it has now been found that the MIP capture coating can be used in a stress sensor. This is in fact very surprising since the MIP matrix first of all needs to be of a sufficiently stiff material to maintain the molecular imprints even when the surface is subjected to stress. Simultaneously, for being used in a stress sensor, the capture coating needs to be sufficiently elastic and flexible to obtain a detectable deformation when subjected to surface stress. According to the present invention it has been found that these two needs actually can be satisfied simultaneously. By selecting the MIP layer to be sufficiently thin, but yet not too thin, the MIP layer can thus surprisingly be used for capture coating in a stress sensor. The stress sensor for detecting a target substance in a fluid according to the invention comprises one sensor unit in the form of a flexible sheet-formed unit.

The sensor unit may in principle have any shape as long as it is sufficiently flexible. In one embodiment, the sensor unit should be capable of deformation under the influence of a deformation force applied as a point force perpendicular to the detecting coating, where the deformation force is 10~3 N or less, such as between 10'3-10"10 N, such as between 10"7-10"5 N, or such as between 10"5-10"3 N. The desired deformation sensitivity (necessary force for obtaining deformation) depends largely on the application of the sensor. In situations where the presence of one or a few target molecules should be detected, the deformation sensitivity should be relatively high, whereas in situations where concentrations of a target substance in a fluid should be detected, the deformation sensitivity may be lower.

Preferably the sensor unit is shaped as a cantilever, a bridge or as a diaphragm. In one embodiment, the sensor further comprises a body which is less flexible than the sensor unit, the at least one sensor unit being linked to said body, the body preferably being essentially non-flexible. ..

In a preferred embodiment of the invention, the sensor comprises one or more cantilevers. The shape and size of the sensor and the size, shape and the number of cantilevers as well as its wiring may e.g. be as disclosed in any one of the patent applications WO 0066266, WO 03071258, WO 03067248, WO 03062135, DK PA 2001 01724, DK PA 2002 00283, DK PA 2002 00125 and DK PA 2002 00195, which with respect to the disclosure concerning structure (shape and size of the sensor and the size, shape and the number of cantilevers as well as its wiring) are hereby incorporated by reference.

In the following the sensor is described with one sensor unit only, but it should be understood that the sensor may have a plurality of sensor units such as up to 300, e.g. up to 100 having equal or different target substance. Also the sensor may comprise a plurality of reference sensor units. The MIP coating can be the same or differ from one sensor unit to another.

In one embodiment, the sensor unit has a cantilever-like shape e.g. as the cantilevers described in WO 03062135. The term 'cantilever' is defined as a sheet-formed unit linked to a substrate along one or two opposite edge lines. The term 'cantilever shape' thus also includes a bridge, as well as a traditional rectangular, triangular or leaf shaped cantilever.

In one embodiment, the cantilever extends in a length between two endings and is linked in both of its endings to form a cantilevered bridge.

In another and preferred embodiment, the cantilever is a traditional rectangular or leaf shaped cantilever linked to one substrate only. In the following this type of cantilever is referred to as cantilever with a free end.

In one embodiment, the cantilever is a flexible sheet-formed unit having an average thickness which is thinner than both of its average length and its average width, said cantilever preferably having a thickness of between 0.05 and 5 μm, such as in the interval of 0.1 μm to 4 μm, such as in the interval of 0.2 μm to 1 μm.

In one embodiment, the cantilever is a flexible sheet-formed unit having an average thickness which is at least 5 times, preferably at least 50 times less than its average width and average length.

The cantilever has a uniform thickness or the thickness may vary. For simple manufacturing, the thickness is essentially uniform.

In one embodiment, the cantilever is essentially plane in non-stressed state, and has a first and a second opposed side, the periphery of the cantilever preferably being essentially rectangular or square formed.

The stress sensor comprises a detecting system capable of detecting changes of stress in the sensor unit. The detector system may in principle be any type of detector system capable of detecting a surface stress of the sensor unit. In one embodiment, the detecting system is an optical read-out system, e.g. as disclosed in WO 99/38007 and US 6289717, the subject matter of which with respect to the detecting system is hereby incorporated by reference.

In one embodiment, the detecting system is a direct electrical read-out system. The detecting system preferably comprises a detector unit preferably selected from the group consisting of a capacitor, a piezo-resistor and a piezoelectric unit, more preferably the detector unit being a piezo-resistor.

In a preferred embodiment, the detecting system comprises a piezo-resistor as the detector unit.

The piezo-resistor incorporated into the sensor unit may e.g. have a thickness in the interval of 10 nm to 500 nm, such as in the interval of 50 nm to 300 nm, such as in the interval of 100 nm to 200 nm, the thickness preferably being uniform.

Useful piezo-resistor includes a piezo-resistor of single crystalline silicon doped with one or more of the ions: boron, arsenic and phosphor.

Such a detecting system is e.g. disclosed in US 6575020, which is hereby incorporated by reference. In an embodiment of the invention, the sensor unit of the sensor is as disclosed in US 6575020 but further provided with a capture coating as disclosed herein.

In one embodiment, it is desired that the detector unit is in physical contact with the sensor unit, preferably the detector unit being incorporated into the sensor unit.

The sensor unit in the form of a piezo-resistor comprises a pair of wires for applying a voltage over the detector unit.

The detecting system preferably comprises a read-out unit connected to the detector unit, the read-out unit preferably being an electrical read-out unit reading out an electrical signal. Such read-out units are in general known to the skilled person. In one embodiment comprising a piezo-resistor where the molecule of interest interacts with the thin MIP layer on the cantilever, a surface stress is generated which stresses the cantilever. The read-out scheme for such sensor can e.g. be obtained by integration of a piezo-resistor in the cantilever using the optical read-out by monitoring the cantilever deflection by a laser beam or by capacitive read-out.

According to the invention the stress sensor comprises at least one sensor unit comprising a capture coating covering at least a part of the surface of the sensor unit. The capture coating comprises a molecular imprinted polymerizate (MIP) for the target substance.

As mentioned above, the molecular imprinted polymerizate (MIP) is disclosed in detail in US 5630978 and US 5959050.

In one embodiment, the sensor unit comprises two major opposite surfaces, the capture coating covering at least a part of one or both of the major opposite surfaces of the sensor unit, preferably the capture coating covering at least 10 %, such as at least 25 %, such as at least 50 %, such as at least 75 % such at essentially all of at least one of the major opposite surfaces of the sensor unit.

For simple production, it is desired in one embodiment that essentially one of the major surfaces of the sensor unit is covered by the capture coating and the other side is essentially free of the capture coating.

The amount of molecular imprints may be equal or it may vary along the capture coating. It has been found that in some situations a too high concentration of molecular imprints may result in a too high 'anti-stress' formed when removing the template substance from the MIP, which may result in a detraction of the capture surface, which can result in a reduced recognizability of the target substance. For assuring recognizability it may therefore be beneficial to vary the concentration of the molecular imprints. The skilled person will also be able to find the desired concentration of molecular imprint by a number of experiments. The monomers for forming the capture coating may in principle be any type of monomers e.g. the monomers disclosed in US 5630978 and US 5959050. In one embodiment, the capture coating is made from one or more monomers which are polymerized to form an imprint for the target substance, the one or more monomers preferably being selected from the group of acrylates, methacrylates, vinyl containing monomer, styrenes, nitriles, siloxanes, silanesbenzenes, pentadienes, pentenes, hexanes, pyridines, urethanes and derivatives of amino acids.

In one embodiment, the molecular imprinted polymerizate (MIP) for the target substance of the capture coating is arranged to form at least one covalent interaction with the target substance, the MIP preferably additionally comprises one or more non-covalent interactions preferably selected from the group of ionic interaction, hydrophobic/hydrophilic interaction, steric interaction, electrostatic interaction and hydrogen bonding interaction.

In one embodiment, the molecular imprinted polymerizate (MIP) for the target substance of the capture coating is arranged to form non-covalent interactions with the target substance, the non-covalent interactions preferably include one, or more of the interactions selected from the group of ionic interaction, hydrophobic/hydrophilic interaction, steric interaction, electrostatic interaction and hydrogen bonding interaction. The MIP may in one embodiment additionally be arranged to form covalent interactions with the target substance. In another preferred embodiment, the MIP is arranged to only comprise one or more of the interactions selected from the group of ionic interaction, hydrophobic/hydrophilic interaction, steric interaction, electrostatic interaction and hydrogen bonding interaction. This embodiment results in a reversible stress sensor which makes on-line monitoring in a system possible.

For arranging the MIP the following two methods are beneficial.

In the one method, a template substance is covalently bound to a polymerizable monomer, and after polymerization the covalent bond is cleaved to release the template substance from the polymeric mold. Using this method, a selected template is attached to a polymerizable moiety using any appropriate method. The polymerizable template should contain a linkage that can be broken to release the template after the MIP is formed, without adversely affecting the MIP. The bond that is cleaved to release the template can optionally provide an additional polar or ionic site for design and 5 imprinting of the mimic.

In another method, polymerizable monomers arrange themselves about a template based on non-covalent interactions (such as ionic, hydrophobic, steric, electrostatic, and hydrogen bonding interactions), and after 10 polymerization, the non-covalently bound template is simply leached out.

The capture coating should preferably be relatively thin, but yet not too thin. The MIP forms a well defined and stable polymeric structure that resembles the outer three-dimensional topology of the target substance, which may e.g. 15 be a relatively small chemical component such as a component of 1000 Dalton or less, a large chemical component, such as a natural biologically active macromolecule or any other chemical component e.g. as mentioned as the target components below. In order to form this three-dimensional topology, and not only a functional surface, the capture coating should not be ~20 too thin. In one embodiment, the capture coating has a thickness of between 0.5 nm and 1000 nm, such as between 1 nm and 500 nm, such as between 10 nm and 100 nm.

In one embodiment, where the sensor unit is a cantilever wherein the 25 cantilever comprises an integrated piezoresistive unit as part of the detecting system, the cantilever preferably comprises a capture coating on one of its major sides, or alternatively on both of its major sides. By having a coating on both sides the cantilever will not necessarily bend but rather be subjected to a stretch, which may have less influence on the shape of the molecular 30 imprints than a bending. Thereby the recognizability of the target substance may be even higher.

In one embodiment, where the sensor unit is a cantilever wherein the cantilever comprises an integrated piezoresistive unit as part of the detecting 35 system, the cantilever comprises a total outer coating of a polymer material, the polymer material of the whole of the outer coating may be made from the same monomers or they may be made from different monomers along the surface of the sensor unit, e.g. so that the capture coating is made from different monomers than the remaining coating.

Thereby the polymer may constitute an insulation against short-circuit, in particular if the stress sensor is for use in a liquid or for detecting a target substance in a liquid.

A part of the outer coating, e.g. one of the major surfaces, may be constituted by the capture coating.

In one embodiment, the capture coating is placed directly in contact with the piezo-resistor. Thereby a high sensitivity may be obtained.

For additional noise reduction the stress sensor may preferably comprise a reference sensor unit which can be used as a common mode rejection filter of noise sources from the environment. The reference sensor unit is linked to the detection system so that the signal from the reference unit is subtracted from the signal from the sensor unit, said subtraction preferably being performed by incorporating the sensor unit and the reference unit in a Wheatstone bridge system, e.g. such as it is disclosed in US 6575020.

The reference unit may preferably be identical with the sensor unit except for the capture coating, thereby the major part of noise can be removed.

In one embodiment, the reference cantilever can be coated with a MIP or with non-imprinted polymer (NIP) and thereby it will experience all the same noise sources as the measurement cantilevers. The noise sources are both mechanical, electrical, chemical and biological including vibrations, changes in the thermal environment, humidity, non-specific bindings etc.

In one embodiment, wherein the reference unit has the same dimensions as the reference unit and is made from the same material, the sensor units capture coating being made from a polymerizate with molecular imprints, the reference unit comprises no capture coating but instead a coating of a polymerizate made from the same monomers as the polymerizate of the capture surface of the sensor unit, but without the molecular imprints.

In one embodiment, wherein the reference unit has the same dimensions as the reference unit and is made from the same material, the sensor units capture coating being made from a polymerizate with molecular imprints for the target substance, the reference unit comprises a fake capture coating of a polymerizate made from the same monomers as the polymerizate of the capture surface of the sensor unit, but with fake molecular imprints, the fake molecular imprint preferably being imprints of another substance than the target substance.

The target substance may be any type of chemical substances for example one or more of the substances selected from the group consisting of one or more binding partners in the form of one or more of the binding components selected from the group comprising one or more bio-molecules of microbial, viral, fungal, plant, animal or human origin, synthetic molecules resembling them, explosives (such as RDX, PETN, TNT), chemical warfare (such as organo-phosphates, organics nerve agents (anticholinesterase), blister .agents (Versicants) and blood agents (cyanogens), alcohols, and drugs, the binding components preferably comprise one or more molecules selected from the group consisting of proteins, glyco proteins, nucleic acids, such as RNA, DNA including cDNA, PNA, LNA, oligonucleotides, peptides, hormones, antigens, antibodies, lipids, sugars, carbohydrates, and complexes including one or more of these molecules, said bio-molecule or molecules preferably being selected from the group consisting of explosives, nucleic acids, antibodies, proteins, protein complexes, enzymes, drugs and receptors.

The stress sensor may comprise one or more fluid chambers, said one or more sensor units in the form of cantilevers partly or totally protruding into said fluid chamber(s) so that a fluid applied in the chamber is capable of coming into contact with part of the surface of the cantilever(s).

The sensor can be used in gas or liquids and be integrated in a micro liquid handling system or placed in a large interaction chamber both having an inlet and an outlet. The sensor can e.g. be used to detect organic, inorganic molecules, proteins, peptides, receptors, ligands, drugs DNA, antibodies, antigens, viruses, pathogens, spores, parasites, explosives, toxins, chemical warfare agents and microorganisms.

With an array of cantilevers it is possible to detect several different analytes in a multi-analyte environment. Furthermore, if a MIP is not 100 % selective towards one analyte, an array of different MIP cantilevers with different selectivities can be used for example in connection with a neural network type of analysis in order to obtain a very high performance sensor with very low rate of false positives.

In order to obtain MIP on the cantilevers, different approaches can be used. Different immobilization methods are known from the literature and not all of the different methods are described here: • Immobilisation of adequate functional groups or free radical initiators onto surfaces can be achieved through for example silane or thiol chemistry, and further polymerisation of the MIP at this level ensures the stable and robust preparation of the cantilevers. • Spin coating the cantilevers with MIP either directly or continuing with in-situ polymerisation of the MIP at this level ensures the stable and robust preparation of the cantilevers. • Ink jet printing of MIP either directly or continuing with in-situ polymerisation of the MIP at this level ensures the stable and robust preparation of the cantilevers. • Molecular imprinting of nano-particles, which are afterwards attached to the cantilever by for example ink-jet printing. • Laser induced grafting of the MIP to the surface. • Photo-induced grafting of the MIP to the surface.

The invention also relates to a method of generating a capture coating for a target substance onto a sensor unit of a stress sensor according to any one of the preceding claims comprising the steps of

i) selecting one or more polymerizable monomers, ii) providing a polymerization reaction mixture comprising the monomer(s), one or more template substance(s) for the target substance and an effective amount of one or more cross-linking agents to provide a stable rigid structure, iii) applying the polymerization reaction mixture onto the surface of the sensor unit, iv) polymerizating the polymerization reaction mixture, and v) removing the template substance.