WO2008019694A2

WO2008019694A2 - Bio surface acoustic wave (saw) resonator design for detection of a target analyte

Info

Publication number: WO2008019694A2
Application number: PCT/DK2007/000379
Authority: WO
Inventors: Peter Warthoe
Original assignee: Atonomics A/S
Priority date: 2006-08-17
Filing date: 2007-08-17
Publication date: 2008-02-21
Also published as: WO2008019694A3

Abstract

The present invention relates to a surface acoustic wave (SAW) resonator unit com prising a plurality of three-dimensional interdigital transducer electrode (IDTE) and reflector micro channel structures located on a piezoelectric substrate surface. The invention relates to a differential immobilization of a molecular recognition component.

Description

Title: BIO SURFACE ACOUSTIC WAVE (SAW) RESONATOR DESIGN FOR DETECTION OF A TARGET ANALYTE

Technical Field The invention relates to the field of sample analysis. In particular, it relates to sample analysis devices such as microsensors based on surface acoustic wave technology and their use. Such microsensors are useful in numerous chemical, environmental and medical applications.

Background

Highly sensitive methods for detection of analyte, such as e.g. biological analyte, continue to be a significant challenge. Frequently, detection methods require processing of multiple samples. In addition, analytical detection methods. should be easy, rapid and reproducible.

Conventional bioanalytical methods have several deficiencies. For example, hybridization of nucleic acid molecules is generally detected by autoradiography or phosphor image analysis, when the hybridization probe contains a radioactive label, or by densitometer, when the hybridization probe contains a label, such as biotin or digoxin. The label can also be recognized by an enzyme-coupled antibody or ligand. Most modern biomolecule detection methods require modification of the molecule, e.g. DNA or RNA or protein, making current detection methods expensive and labor intensive.

Acoustic wave sensor technology has shown broad application in detecting materials. Acoustic wave sensors detect materials by generating and observing an acoustic wave. As the acoustic wave propagates through or on the surface of the material, any changes to the characteristics of the propagation path affect the velocity and/or amplitude of the wave. The amplitude, frequency and/or phase characteristics of the sensor can be measured and correlated to a corresponding physical quantity.

Several different types of acoustic wave devices have been developed, but all of them have only limited success in measuring soluble or biological samples. Bulk acoustic waves (BAW) propagate through a medium. The most commonly used BAW devices are the thickness shear mode (TSM) resonator. The most common types are quartz crystal microbalances and the shear-horizontal acoustic plate mode (SH-APM) sensor. Conversely, waves that propagate on the surface of the substrate are known as surface waves. The most widely used surface wave devices are the surface acoustic wave sensor and the shear-horizontal surface acoustic wave (SH-SAW) sensor, also known as the surface transverse wave (STW) sensor. All acoustic wave sensors will function in gaseous or vacuum environments, but very few of them will operate efficiently, when they are in contact with liquids.

Of the known acoustic sensors for liquid sensing, the Love wave sensor, a special class of the shear-horizontal SAW, has the highest sensitivity. To make a Love wave sensor, a dielectric wave guide coating is placed on a SH-SAW device such that the energy of the shear-horizontal waves is focused in that coating. A biorecognition coat- ing is then placed on the wave guide coating, forming the complete biosensor. Successful detection of anti-goat IgG in the concentration range of ng/ml using a 110 MHz YZ-cut SH-SAW with a polymer Love wave guide coating has been achieved [E. Gizeli et al. 1997. "Antibody Binding to a Functionalized Supported Lipid Layer: A Direct Acoustic Immunosensor," Anal Chem, Vol. 69:4808-4813].

A comparison between different SAW sensors has recently been described [Bio- molecular Sensors, Eds. Electra Gizeli and Christoffer R. Lowe (2002)]. Gizeli and Lowe describe a 124 MHz Love wave sensor having a sensitivity of 1.92 mg/cm2. The use of SAW sensors for detection of biological compounds has been reported in, for example, US 5,478,756, WO9201931 and WO03019981 , each of which being incorporated in the present application by reference in its entirety.

Conventional SAW devices are a poor choice for liquid detection, as the vertical component of the propagating wave is suppressed by the liquid-air barrier. One acoustic wave sensor that functions in liquids is a shear-horizontal SAW sensor. If the cut of the piezoelectric crystal material is rotated appropriately, waves propagate horizontally and parallel to a liquid surface. This reduces loss dramatically, when liquids come into contact with the propagating medium, allowing the SH-SAW sensor to operate as a biosensor. Many efforts at detecting liquid solution analyte, such as biological molecules, have focused on defining the interaction between the acoustic wave and the properties of the solid/liquid interface, as well as on designing higher frequency SAW devices operating in the GHz range.

The present application provides a new solution for special designed bio SAW resona- tor devices to measure test analytes, including biomolecules, in a sample. Saw devices of the two-dimensional type, where for example reflector structures are absent, containing molecular recognition components immobilized over the entire surface of the recognition element have been described. However these devices suffer from the drawback of poor detection capabilities in several applications.

Accordingly, it is an object of the present invention to obtain a SAW device of the resonator type with improved detection capabilities.

Disclosure of the Invention The inventors of the present invention surprisingly found that differential placement of the molecular recognition element only on selected parts of the resonator unit lead to a significantly altered signal, thereby providing benefits in terms of sensitivity and intensity of the signal. This leads to improvements of the resulting device in term of specificity and limit of detection of specific analytes.

Accordingly, in one aspect, the present invention relates to a surface acoustic wave (SAW) resonator unit comprising:

(a) a piezoelectric substrate,

(b) at least one interdigital transducer electrode (IDTE) structure, (c) at least one reflector structure, and

(d) at least one molecular recognition component, wherein three-dimensional micro channels are formed within the IDTE structure(s), and wherein three-dimensional micro channels are formed within the reflector structure(s), and in which the at least one molecular recognition component is immobilized only in selected parts of the surface acoustic wave resonator unit, whereby at least one part of the resonator unit does not contain immobilized molecular recognition components.

In a second aspect the invention relates to a microsensor comprising at least one surface acoustic wave (SAW) resonator unit comprising: (a) a piezoelectric substrate,

(b) at least one interdigital transducer electrode (IDTE) structure,

(c) at least one reflector structure, and

In a third aspect the invention relates to a handheld device for detecting target analytes comprising the microsensor.

In a fourth aspect the present invention relates to the use of the microsensor for detecting a target analyte in a sample.

Brief Description of the Drawings

The invention is explained in detail below with reference to the drawings, in which

Fig. 1 illustrates a SAW device comprising, on separate piezoelectric substrates, two SAW resonator units indicated generally by the reference numerals 1 and 2. Each SAW resonator unit consists of one IDTE region (6, 7) and two reflector regions (5, 8). Micro channels (4) are located between the IDTE structures (3). Identical micro channels are also located between the reflector structures (5, 8). The SAW resonator unit (1 ) has been immobilized with molecular recognition components on and between the IDTE structures (7), whereas the identical IDTE structures (6) on SAW resonator units (2) do not have any molecular recognition component immobilized. The SAW resonator unit (2) has been immobilized with molecular recognition components on and between the reflector structures (5), whereas the identical reflector structures (8) on SAW resonator units (1) do not have any molecular recognition component immobilized.

Fig. 2 illustrates two SAW resonator units (1 , 2) on the same piezoelectric substrate, otherwise identical to Fig. 1.

Fig. 3 illustrates a SAW device comprising, on separate piezoelectric substrates, two SAW resonator units indicated generally by the reference numerals 1 and 2. Each SAW resonator unit consists of one IDTE part (6, 7) and two reflector parts (5, 8). The micro channels (4) are located between the IDTE (3). Similar micro channels are also located between the reflector structures (5, 8). The SAW resonator unit (1 ) has been immobilized with molecular recognition components only in the channels between the IDTE structures (7), whereas the identical IDTE structures (6) on SAW resonator units (2) do not have any molecular recognition component immobilized. The SAW resonator unit (2) has been immobilized with molecular recognition components only in the channels between the reflector structures (5), whereas the identical reflector structures (8) on SAW resonator units (1 ) do not have any molecular recognition component immobilized. On a subpart of the reflector structures (8) it is shown that the immobilization of molecular recognition components only takes place in the channels between the reflector structures.

Fig. 4 illustrates two SAW resonator units (1 , 2) on the same piezoelectric substrate, otherwise identical to Fig. 3.

Fig. 5 illustrates a SAW device comprising, on separate piezoelectric substrates, two SAW resonator units indicated generally by the reference numerals 1 and 2. Each

SAW resonator unit consists of one IDTE part (6, 7) and two reflector parts (5, 8). The micro channels (4) are located between the IDTE (3) . Similar micro channels are also located between the reflector structures (5, 8). The SAW resonator unit (1) has been immobilized with molecular recognition components on the entire three-dimension sur- face including reflector structures (8), IDTE structures (7) and micro channel structures.

The SAW resonator unit (2) has no molecular recognition component immobilized on the three-dimensional surface (10).

Fig. 6 illustrates two SAW resonator units (1 , 2) on the same piezoelectric substrate, otherwise identical to Fig. 5.

Fig. 7 illustrates a SAW device comprising, on separate piezoelectric substrates, two SAW resonator units indicated generally by the reference numerals 1 and 2. Each SAW resonator units consists of one IDTE part (6, 7) and two reflector parts (5, 8). The micro channels (4) are located between the IDTE (3). Similar micro channels are also located between the reflector structures (5, 8). The IDTE structures (7) on SAW resonator unit (1 ) have been immobilized with molecular recognition components (7). The IDTE structures (6) on SAW resonator unit (2) have a non-specific IgG molecule immobilized not capable of binding the analyte as reference.

Fig. 8 illustrates a SAW device comprising, on separate piezoelectric substrates, two SAW resonator units indicated generally by the reference numerals 1 and 2. Each SAW resonator unit consists of one IDTE part (6, 7) and two reflector parts (5, 8). The micro channels (4) are located between the IDTE (3). Similar micro channels are also located between the reflector structures (5, 8). The reflector structures (8) on SAW resonator unit (1 ) have been immobilized with molecular recognition components (8). The reflector structures (5) on SAW resonator unit (2) have a non-specific IgG molecule immobilized not capable of binding the analyte as reference.

Fig. 9 illustrates one SAW resonator unit indicated generally by the reference numeral 1. The SAW resonator units consist of one IDTE part (9) and two reflector parts (6, 8).

The micro channels (4) are located between the IDTE (3). Identical micro channels are also located between the reflector structures (6, 8). The SAW resonator unit (1) has been immobilized with molecular recognition components on one half of the entire three-dimension surface (10) including reflector structures (8), IDTE structures (2) and micro channel structures (3). The second half of the SAW resonator unit (7) does not have any molecular recognition component immobilized.

Detailed Description of the Invention Definitions "Binding event" as used in this application means the binding of the target analyte to the molecular recognition component immobilized in the three-dimensional channel structure surface of the SAW sensor.

"Homogeneous set" as used in this application means a region of the three- dimensional sensor surface, where only IDTE or reflector structures are present, but no mixture of structures.

"Differentially immobilized" as used in this application means that the at least one molecular recognition component is immobilized onto certain IDTE and/or resonator struc- tures of at least one first unit, and optionally onto certain other IDTE and/or resonator structures of at least one second unit, thereby generating a set of units having the at least one molecular recognition component immobilized differently on each unit.

"A set of units" as used in this application means that a set is made by at least two units. It may comprise more than two such as three, four, five, six, seven, eight etc.

"Molecular recognition component" or "molecular recognition element" as used in this application means a species, which is capable of binding a target species or analyte. Suitable examples of "molecular recognition components" are nucleic acids, nucleotide, nucleoside, nucleic acids analogues such as PNA and LNA molecules, proteins, peptides, antibodies including IgA, IgG, IgM, IgE, enzymes, enzymes cofactors, enzyme substrates, enzymes inhibitors, receptors, ligands, kinases, Protein A, Poly U, Poly A, Poly lysine, triazine dye, boronic acid, thiol, heparin, polysaccharides, coomassie blue, azure A, metal-binding peptides, sugar, carbohydrate, chelating agents, prokaryotic cells and eukaryotic cells. The molecular recognition element may be immobilized chemically to the substrate or may be immobilized in a gel, such as a hydrogel, which is then immobilized on the relevant structure.

"Immobilization matrix" as used in this application means a fluid or semi-fluid compartment capable of containing the "molecular recognition component" or "molecular recognition element", e.g. a gel or hydrogel. Preferably, the "immobilization matrix" changes properties after binding or capturing the analyte by the "molecular recognition component".

Surface Acoustic Wave Sensors

The microsensors disclosed in this application comprise at least one surface acoustic wave sensor. A surface acoustic wave sensor comprises a piezoelectric layer, or piezoelectric substrate, an input and output transducer. A surface acoustic wave is generated within the piezoelectric layer and is detected by interdigitated electrodes. As described in more detail below, binding events that alter the surface of the surface acoustic wave sensor can be detected as a change in a property of the propagating surface acoustic wave. Surface acoustic wave sensors are described in US 5,130,257, 5,283,037 and 5,306,644; F. Josse, et. al. "Guided Shear Horizontal Surface Acoustic Wave Sensors for Chemical and Biochemical Detection in Liquids," Anal. Chem. 2001 , 73, 5937; and W. Welsch, et. al., "Development of a Surface Acoustic Wave Im- munosensor," Anal. Chem. 1996, 68, 2000-2004, each of which hereby expressly be- ing incorporated in its entirety by reference.

Acoustic wave devices are described by the mode of wave propagation through or on a piezoelectric substrate. Acoustic waves are distinguished primarily by their velocities and displacement directions. Many combinations are possible, depending on the mate- rial and boundary conditions. The interdigital transducer electrode (IDTE) of each sensor provides the electric field necessary to displace the substrate and thus form an acoustic wave. The wave propagates through the substrate, where it is converted back to an electric field at the IDTE at the opposing electrode. Transverse or shear waves have particle displacements that are normal to the direction of wave propagation and which can be polarized so that the particle displacements are either parallel to or normal to the sensing surface. Shear-horizontal wave motion signifies transverse dis- placements polarized parallel to the sensing surface; shear-vertical motion indicates transverse displacements normal to the surface.

"Surface acoustic wave sensor" or "surface acoustic wave device" as used in this appli- cation means any device that operates substantially in the manner described above. In some embodiments, "surface acoustic wave sensor" refers to both surface transverse wave devices, where the surface displacement is perpendicular to the direction of propagation and parallel to the device surface, as well as to surface acoustic wave sensors, where at least a portion of the surface displacement is perpendicular to the device surface. While surface transverse wave devices generally have better sensitivity in a fluid, it has been shown that sufficient sensitivity may also be achieved, when a portion of the surface displacement is perpendicular to the device surface. See, for example, M. Rapp, et al. "Modification of Commercially Available LOW-LOSS SAW devices towards an immunosensor for in situ Measurements in Water" 1995 IEEE International Ultrasonics Symposium, Nov. 7-10, 1995, Seattle, Wash.; and N. Barie, et al., "Cova- lent bound sensing layers on surface acoustic wave biosensors," Biosensors & Bio- electronics 16 (2001) 979, all of which being expressly incorporated herein by reference.

The preferred saw device according to the invention is a SAW sensor of the resonator type (a SH-saw device).

The sensors are made by a photolithographic process. Manufacturing begins by carefully polishing and cleaning the piezoelectric substrate. A coating layer made from metal, such as e.g. gold (Au), silver (Ag), SiO2, aluminium (Al) or any kind of polymer, is then deposited uniformly onto the substrate. The device is spin-coated with a photoresist, which is hardened by baking. It is then exposed to UV light through a mask with opaque areas corresponding to the areas to be metalized on the final device. The UV- exposed areas undergo a chemical change that allows them to be removed with a de- veloping solution. Finally, the remaining photoresist is removed. The pattern of metal remaining on the device is called an interdigital transducer, interdigital electrode or in- terdigital tranducer electrode (IDTE). By changing the length, width, position and thickness of the IDTE, the performance of the sensor can be optimized.

Coating of a SAW sensor of the BAW-type, i.e. a flat sensor without resonators, employing the entire sensor surface has been described. The inventor has discovered that change of frequency of a SAW sensor of the resonator type or SAW filter type highly depends on which structure is involved in the binding event. If the binding event takes place in the IDTE region, the frequency will most likely decrease, but can also increase under other circumstances. If the binding event takes place in the reflector region, the frequency can either decrease or increase depending on experimental setup and re- flector design.

The signal generated by the microsensor of the present invention is dependent on stiffness changes in the bio-film in the three-dimensional micro channels in the IDTE and the reflector structures. The signal may either be an increase or a decrease in the fre- quency of the SAW resonator unit depending on the setup (see: US application 20060024813 by the same inventor as the present application).

Accordingly, the present invention relates to a differential coating of an attachment layer on either the IDTE structures or the reflector structures or on both structures and the three-dimensional channels between said structures. Only the differentially coated attachment layer will bind the molecular recognition component and thereby the target analyte. Molecular recognition molecules may be attached directly to self-assembled monolayers. For example, when gold electrodes are employed, a DNA probe molecule may be attached using a SH group on the 5' of the DNA using self-assembled monolayers as known in the art.

In a first embodiment the invention relates to a surface acoustic wave (SAW) resonator unit comprising: (a) a piezoelectric substrate, (b) at least one interdigital transducer electrode (IDTE) structure,

(c) at least one reflector structure, and

(d) at least one molecular recognition component, wherein three-dimensional micro channels are formed within the IDTE structure of (b), and wherein three-dimensional micro channels are formed within the reflector structure of (c), and in which the at least one molecular recognition component is immobilized only in selected parts of the surface acoustic wave resonator unit, whereby at least one part of the resonator unit does not contain immobilized molecular recognition components.

Thereby a significant alteration in signal propagation and detection is achieved. The selected parts of the surface acoustic wave resonator unit which may comprise the at least one immobilized molecular recognition component are (1 ) the IDTE structure^), (2) the reflector structure(s) or (3) a selected combination of one IDTE structure and one reflector structure of the unit. In a most preferred embodiment the selected parts of the surface acoustic wave resonator unit which comprises the at least one immobilized molecular recognition component are the IDTE structure(s) of the at least one resonator unit.

In one embodiment the at least one immobilized molecular recognition component is found only, or substantially only, in the micro channels of (1) the IDTE structure(s), (2) the reflector structure(s) or (3) a selected combination of one of the IDTE structures and the micro channels of one of the reflector structures.

SAW devices are very small and selective placement of immobilized molecular recogni- tion components only in the micro channels is technically very challenging. Consequently, in this context, the term "substantially only in the micro channels" means that the device is manufactured with the intent of only placing the at least one immobilized molecular recognition component in the respective micro channels. In other words, the term "substantially only in the micro channels " means that more than 50%, such as more than 60%, or more than 70%, or more than 80%, or more than 90% of the immobilized molecular recognition components is found in the respective micro channels of the resonation unit.

The inventors found that immobilization of molecular recognition on the reflector struc- tures lead to a loss of signal due to a decreased reflection of the acoustic waves. Accordingly, in a preferred aspect of the invention the at least one molecular recognition component is immobilized only on the IDTE structure(s) of the resonator unit(s).

Preferably, in the resonator unit according to the invention, at least 25% of the total of the reflector structures is free of immobilized molecular recognition components and at least 25% of the total of the IDTE structures comprises immobilized molecular recognition components.

The easiest mode of fabrication of the resonator units of the invention is to cover the entire relevant structure with the molecular recognition element. Accordingly, in one embodiment of the invention the at least one molecular recognition component is im- mobilized both in the micro channels as well as on top of the respective structures, preferably the IDTE structure(s).

However, the inventors found that immobilization of molecular recognition elements only, or substantially only, in the micro channels of the respective structures lead to a more reproducible and increased signal, with a minimal use of recognition elements.

Without wishing to be bound by theory, it is believed that an increased viscosity of the tree-dimensional micro channel space only produces the significant alteration of the signal. Accordingly, in one aspect of the invention the at least one molecular recogni- tion component is immobilized only, or substantially only, in the micro channels of the respective structures, preferably the IDTE structure(s).

A change in viscosity on binding between the analyte and the molecular recognition element was found to be best achieved, if the molecular recognition element is immobi- lized in an immobilization matrix. Accordingly, in one aspect of the invention the at least one molecular recognition component is immobilized in an immobilization matrix. In a preferred embodiment hereof the immobilization matrix comprising the molecular recognition component changes viscosity in response to binding between the analyte and the molecular recognition component.

In order to obtain the best results in terms of altered signal upon binding between analyte and molecular recognition element, the resonator unit should contain at least two adjacent IDTEs having a height from 10 nm to 1 micron and where the micro channel between said adjacent IDTEs has a width from 100 nm to 10 microns.

In order to obtain the best results in terms of altered signal upon binding between analyte and molecular recognition element, the resonator unit should contain at least two adjacent reflectors having a height from 10 nm to 1 micron and where the micro channel between said adjacent reflectors has a width from 100 nm to 10 microns.

In order to obtain the best results in terms of altered signal upon binding between analyte and molecular recognition element the resonator unit should contain at least two adjacent I DTE/reflectors junctions having a height from 10 nm to 1 micron and where the micro channel between said adjacent structures has a width from 100 nm to 10 mi- crons. The molecular recognition component is preferably selected from the group consisting of nucleic acids, nucleotide, nucleoside, nucleic acids analogues such as PNA and LNA molecules, proteins, peptides, antibodies including IgA, IgG, IgM, IgE, enzymes, enzymes cofactors, enzyme substrates, enzymes inhibitors, receptors, ligands, kinases, Protein A, Poly U, Poly A, Poly lysine, triazine dye, boronic acid, thiol, heparin, polysaccharides, coomassie blue, azure A, metal-binding peptides, sugar, carbohydrate, chelating agents, prokaryotic cells and eukaryotic cells.

The resonator units according to the invention may be used in a microsensor suitable for detecting an analyte species from a sample.

Preferably, the microsensor comprises at least one set of surface acoustic wave (SAW) resonator units according to the invention.

Accordingly, the present invention relates to a microsensor for measuring a signal upon detection of a target analyte in a sample, said microsensor comprising at least one set of surface acoustic wave (SAW) resonator units, each unit comprising:

(a) a piezoelectric substrate,

(d) at least one molecular recognition component, wherein three-dimensional micro channels are formed within the IDTE structure, and wherein three-dimensional micro channels are formed within the reflector structure, and in which the at least one molecular recognition component is immobilized only in se- lected parts of the set of surface acoustic wave resonator units, whereby at least one part of the set of resonator units does not contain immobilized molecular recognition components.

The selected parts of the set of surface acoustic wave resonator units which may com- prise the at least one immobilized molecular recognition component are (1 ) the IDTE structure(s), (2) the reflector structure(s) or (3) a selected combination of the IDTE structures of one of the units constituting the set of units and the reflector structures of the other of the units constituting the set of units. In a most preferred embodiment the selected parts of the set of surface acoustic wave resonator units which comprises the at least one immobilized molecular recognition component are the IDTE structure(s) of at least one, and preferably both, of the resonator units. In one embodiment the at least one immobilized molecular recognition component is found only, or substantially only, in the micro channels of (1) the IDTE structure(s), (2) the reflector structure(s) or (3) a selected combination of the IDTE structures of one of the units constituting the set of units and the micro channels of the reflector structures of the other of the units constituting the set of units.

In order to achieve a signal that can be used to calculate the amount of analyte, the signal must be compared to a reference (background) signal. Thus, in a preferred embodiment the microsensor further comprises at least one reference surface acoustic wave (SAW) resonator unit that does not comprise immobilized molecular recognition components. Preferably, the microsensor comprises at least one set of surface acoustic wave (SAW) resonator units that do not comprise immobilized molecular recognition components. Alternatively, the reference can be a measure of a sample solution that does not comprise the target analyte. The difference between the signal of the refer- ence micro channels and the sensor micro channels determines the presence of the target analyte.

In one embodiment of the invention the at least one molecular recognition component is immobilized only on the IDTE structure of a first unit and on the reflector of a second unit.

In one embodiment of the invention the units of a set are placed on the same piezoelectric substrate.

In one embodiment of the invention the units of a set are placed on separate piezoelectric substrates.

SAW sensors are small sensors making the technology suitable for use in handheld devices. Accordingly, the invention further relates to a handheld device for detecting target analytes comprising the microsensor according to the above.

In a preferred aspect the at least one molecular recognition component is immobilized only, or substantially only, in the micro channels of the IDTE and/or the reflector structures.

The microsensor according to the invention is suitable for multiplex detection of two or more target analytes. Accordingly, in one aspect of the invention the microsensor com- prises different molecular recognition components immobilized on the two units of a pair.

The relative small SAW device according to the invention makes it suitable for use in clinics, operated on site by clinical staff. Accordingly, the microsensor is ideal for use in connection with detection of analytes from samples selected from the group consisting of blood, serum, plasma, faeces, spinal core fluids, urine, smears and saliva.

One embodiment of the invention relates to the use of the microsensor according to the invention for measuring a signal upon detection of a target analyte in a sample.

Preferably, the target analyte is selected from the group consisting of Troponin I₁ Troponin T, BNP, an H-FABP, an allergen and IgE.

In one aspect the present invention relates to a microsensor for measuring a signal upon detection of a target analyte in a sample, said microsensor comprising at least one set of surface acoustic wave (SAW) resonator units, each unit comprising: a piezoelectric substrate; at least one interdigital transducer electrode (IDTE) structure; at least one reflector structure; and at least one molecular recognition component; wherein three-dimensional micro channels are formed within the IDTE structure, and wherein three-dimensional micro channels are formed within the reflector structure, and in which at least one molecular recognition component is immobilized only in and/or on the IDTE structure of at least a first unit and in and/or on the reflector structure of at least a second unit, thereby forming a set of SAW resonator units which is differentially immobilized with the at least one molecular recognition component. Such an embodiment is shown in Fig. 1 of the present application. Preferably, the molecular recognition component is only immobilized in the micro channels of the reflector structures and/or the micro channels of the IDTE structure respectively. Such an embodiment is shown in Fig. 3.

In another aspect the present invention relates to a microsensor for measuring a signal upon detection of a target analyte in a sample, said microsensor comprising at least one set of surface acoustic wave (SAW) resonator units, each unit comprising: a piezoelectric substrate; at least one interdigital transducer electrode (IDTE) structure; at least one reflector structure; and optionally, at least one molecular recognition component; wherein three-dimensional micro channels are formed within the IDTE structure, and wherein three-dimensional micro channels are formed within the reflector structure, and in which at least one molecular recognition component is immobilized only in and/or on both the IDTE structure and the reflector structure of at least one first unit, whereas the micro channels of both the IDTE structure and the reflector structure of at least one second unit do not comprise said molecular recognition component, thereby forming a set of SAW resonator units which is differentially immobilized with the at least one molecular recognition component. Such an embodiment is shown in Fig. 5. Preferably, the molecular recognition component is only immobilized in the micro channels of the reflector structure and/or the micro channels of the IDTE structure respectively.

In yet another aspect the present invention relates to a surface acoustic wave (SAW) resonator unit comprising: a piezoelectric substrate; at least one interdigital transducer electrode (IDTE) structure; at least two reflector structures; and at least one molecular recognition component; wherein three-dimensional micro channels are formed within the IDTE structure and wherein three-dimensional micro channels are formed within the reflector structure, and in which at least one molecular recognition component is immobilized only in or on selected parts of the surface acoustic wave resonator unit, whereby at least a part of the resonator unit does not contain immobilized molecular recognition components. Such an embodiment is shown in Fig. 9. Preferably, the molecular recognition component is only immobilized in the IDTE structure. Preferably, the molecular recognition component is only immobilized in the micro channels of the relevant structure.

The selected parts of the surface acoustic wave resonator unit which may comprise the at least one immobilized molecular recognition component are (1) the IDTE structure, (2) the reflector structure or (3) a selected combination of a part of the IDTE structure and part of the reflector structure.

In a most preferred embodiment the selected parts of the resonator unit which comprises the at least one immobilized molecular recognition component is the IDTE struc- ture of the resonator units.

In one embodiment at least one part of the reflector structure does not contain immobilized molecular recognition components.

In one embodiment the at least one immobilized molecular recognition component is found only, or substantially only, in the micro channels of (1 ) the IDTE structure, (2) the reflector structure or (3) a selected combination of a part of the IDTE structure and a part of the reflector structure.

The invention relates to a microsensor for detecting the presence of a target analyte in a test sample solution comprising at least one surface acoustic wave (SAW) resonator units comprising: a piezoelectric substrate; a plurality of interdigital transducer electrode (IDTE) structures and reflector structures on a surface of said substrate; wherein three-dimensional micro channels are formed between said IDTE and reflector structures; having at least one molecular recognition component differentially immobilized in and/or on said IDTE structures or reflector structures. Preferably, the at least one molecular recognition component is immobilized substantially only in said three- dimensional micro channels.

The invention further relates to a production method, where it is possible to direct a mi- crosensor having a sensing and a reference structure on the same microsensor surface. By designing a differential attachment layer coating strategy, it is possible to have an attachment layer coated onto only part(s) of the IDTE's and/or reflectors' three- dimensional structures, whereas other parts of the IDTE's and reflectors' three- dimensional structures are without attachment layer coating serving as reference for the coated IDTE and reflectors structures.

The invention further relates to a device, where only the sets of IDTE structures comprise immobilized molecular recognition components and where reference reflector structures are present on the same piezoelectric substrate.

It has been observed that the amplitude of the SAW resonator unit should be adjusted for optimal conditions for at least one molecular recognition component to react with the target analyte in the test sample. If the amplitude is too high, inconsistent results can be obtained due to non-optimal conditions for molecular recognition compo- nent/analyte interaction.

The present invention is directed to microsensors and their use for detecting a target analyte in a sample. The microsensors comprise a molecular recognition component differentially immobilized on homogeneous sets of IDTE and reflector structures on the three-dimensional surface of a SAW sensor. When the molecular recognition component binds a target analyte, a change in phase, amplitude or frequency of the surface wave is observed, thereby determining the presence of the target analyte in the sam- pie. The acoustic wave sensor disclosed herein thus provides a detection method readily adaptable to detecting liquid-soluble analytes, including biological molecules such as e.g. nucleic acids and proteins, at high sensitivity and in the absence of labelling.

One embodiment of the invention relates to a microsensor for measuring a signal upon detection of a target analyte in a sample, said microsensor comprising at least one set of surface acoustic wave (SAW) resonator units, each unit comprising:

(a) a piezoelectric substrate,

(d) at least one molecular recognition component, wherein three-dimensional micro channels are formed within the IDTE structure, and wherein three-dimensional micro channels are formed within the reflector structure, and in which at least one molecular recognition component is immobilized only in or on the IDTE structure of at least one first unit and in or on the reflector structure of at least one second unit, thereby forming a set of SAW resonator units which is differentially immobilized with the at least one molecular recognition component.

In one embodiment thereof both units of a pair are placed on the same piezoelectric substrate. In one embodiment thereof both units of a pair are placed on separate piezoelectric substrates.

(a) a piezoelectric substrate,

(b) at least one interdigital transducer electrode (IDTE) structure,

(c) at least one reflector structure, and

(d) at least one molecular recognition component, wherein three-dimensional micro channels are formed within the IDTE structure, and wherein three-dimensional micro channels are formed within the reflector structure, and in which at least one molecular recognition component is only immobilized in the micro channels of the IDTE structure of at least one first unit and in the micro channels of the reflector structure of at least one second unit, thereby forming a set of SAW resonator units which is differentially immobilized with the at least one molecular recognition component. In one embodiment thereof both units of a pair are placed on the same piezo- electric substrate. In one embodiment thereof both units of a pair are placed on separate piezoelectric substrates.

(a) a piezoelectric substrate,

(b) at least one interdigital transducer electrode (IDTE) structure,

(c) at least one reflector structure, and (d) optionally, at least one molecular recognition component, wherein three-dimensional micro channels are formed within the IDTE structure, and wherein three-dimensional micro channels are formed within the reflector structure, and in which at least one molecular recognition component is only immobilized in or on both the IDTE structure and the reflector structure of at least one first unit, whereas both the IDTE structure and the reflector structure of at least one second unit do not comprise said molecular recognition component, thereby forming a set of SAW resonator units which is differentially immobilized with the at least one molecular recognition component. In one embodiment thereof both units of a pair are placed on the same piezoelectric substrate. In one embodiment thereof both units of a pair are placed on separate piezoelectric substrates.

One embodiment of the invention relates to a microsensor for measuring a signal upon detection of a target analyte in a sample, said microsensor comprising at least one set of surface acoustic wave (SAW) resonator units, each unit comprising: (a) a piezoelectric substrate,

(b) at least one interdigital transducer electrode (IDTE) structure,

(c) at least one reflector structure, and

(d) optionally, at least one molecular recognition component, wherein three-dimensional micro channels are formed within the IDTE structure, and wherein three-dimensional micro channels are formed within the reflector structure, and in which at least one molecular recognition component is only immobilized in the micro channels of both the IDTE structure and the reflector structure of at least one first unit, whereas the micro channels of both the IDTE structure and the reflector structure of at least one second unit do not comprise said molecular recognition component, thereby forming a set of SAW resonator units which is differentially immobilized with the at least one molecular recognition component. In one embodiment thereof both units of a pair are placed on the same piezoelectric substrate. In one embodiment thereof both units of a pair are placed on separate piezoelectric substrates.

One embodiment of the invention relates to a surface acoustic wave (SAW) resonator unit comprising:

(a) a piezoelectric substrate,

(b) at least one interdigital transducer electrode (IDTE) structure,

(c) at least one reflector structure, and

(d) at least one molecular recognition component, wherein three-dimensional micro channels are formed within the IDTE structure, and wherein three-dimensional micro channels are formed within the reflector structure, and in which the at least one molecular recognition component is immobilized only in and/or on selected parts of the surface acoustic wave resonator unit, whereby at least one part of the resonator unit does not contain immobilized molecular recognition compo- nents. In one embodiment hereof, at least one part of the IDTE structure does not contain immobilized molecular recognition components. In another embodiment hereof, at least one part of the reflector structure does not contain immobilized molecular recognition components. Preferably, at least 25% of the IDTE structure and/or the reflector structure is free of immobilized molecular recognition components and at least 25% of the IDTE structure and/or the reflector structure comprises immobilized molecular recognition components.

One embodiment of the invention relates to a surface acoustic wave (SAW) resonator unit comprising: (a) a piezoelectric substrate,

(b) at least one interdigital transducer electrode (IDTE) structure,

(c) at least two reflector structures, and

(d) at least one molecular recognition component, wherein three-dimensional micro channels are formed within the IDTE structure, and wherein three-dimensional micro channels are formed within the reflector structure, and in which at least one molecular recognition component is immobilized only in or on both the IDTE structure and the reflector structure of one part of the unit, thereby forming a

SAW resonator unit which is differentially immobilized with the at least one molecular recognition component. Preferably, at least 25% of the IDTE structure and/or the reflec- tor structure is free of immobilized molecular recognition components and at least 25% of the IDTE structure and/or the reflector structure comprises immobilized molecular recognition components. The invention further relates to a device comprising the above mentioned resonator unit.

One embodiment of the invention relates to a microsensor for measuring a signal upon detection of a target analyte in a sample, said microsensor comprising at least one surface acoustic wave (SAW) resonator unit comprising:

(a) a piezoelectric substrate,

(b) at least one interdigital transducer electrode (IDTE) structure, (c) at least two reflector structures, and

(d) at least one molecular recognition component, wherein three-dimensional micro channels are formed within the IDTE structure and wherein three-dimensional micro channels are formed within the reflector structure, and in which at least one molecular recognition component is only immobilized in the micro channels of both the IDTE structure and the reflector structure of one part of the unit, thereby forming a SAW resonator unit which is differentially immobilized with the at least one molecular recognition component.

It is clear to the skilled person that units described in the embodiments of the present invention supra may be used in various combinations to generate microsensors.

One embodiment of the invention relates to the microsensors, wherein the at least one molecular recognition component is immobilized both in the micro channels as well as on top of the IDTE and the reflector structures.

One embodiment of the invention relates to the microsensors, wherein the signal changes in response to a change in a liquid/solid volume in said three-dimensional micro channels of the IDTE and the reflector structure.

Input and Output Transducer(s)

The input and output transducers are preferably interdigital transducers. Generally, there are two interdigital transducers. Each of the input and output transducers com- prises two electrodes arranged in an interdigitated pattern. A voltage difference applied between the two electrodes of the input transducer results in the generation of a sur- face acoustic wave in the piezoelectric substrate. The electrodes generally may comprise any conductive material, with aluminium or gold being preferred.

The electrode(s) may take any conventional form, but are preferably photolithographi- cally deposited on the surface as elongate regions of metallisation transverse to the direction of propagation of a wave along the surface of the support. The elongate metallized regions preferably have a width and spacing of the same order of magnitude.

The width of the electrode is typically between 1 and 40 microns, preferably between 10 and 20 microns. In certain embodiments, the width is greater than or equal to 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1 micron, 2 microns, 3 microns, 4 microns, 5 microns, 7.5 microns, 10 microns, 15 microns, 20 microns, 25 microns, 30 microns, 35 microns, 40 microns, 45 microns, 50 microns, 60 microns, 70 microns, 80 microns or 90 microns.

The space between the electrodes can be equal to or less than 100 microns, 90 microns, 80 microns, 70 microns, 60 microns, 50 microns, 45 microns, 40 microns, 35 microns, 30 microns, 25 microns, 20 microns, 15 microns, 10 microns, 7.5 microns, 5 microns, 4 microns, 3 microns, 2 microns 1 microns, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, or 75 nm. It should be noted that the spacing varies inversely with the frequency of the device.

In certain embodiments, the height of the electrodes is the same as the width of the electrodes. In other embodiments, the height of the electrodes is greater than or equal to 10 nm, 20 nm, 30 nm, 40 nm, 50 nm, 75 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, or 900 nm.

In some embodiments, the depth of the space between the electrodes can be less than or equal to 1 micron, 900 nm, 800 nm, 700 nm, 600 nm, 500 nm, 400 nm, 300 nm, 200 nm, 100 nm, 75 nm, 50 nm, 40 nm, 30 nm, or 20 nm.

In an alternative embodiment there is a single interdigital transducer. In this embodiment the single interdigital transducer serves both as input and output transducer. In embodiments employing a single interdigital transducer acting as both input and output transducer, a reflector structure is generally provided to generate one or more resonances within the SAW sensor. The reflector structure may, for example, be a thin film grating. The grating may comprise aluminium or another conductive material. The gen- erated resonances can be detected, for example, by measuring the power dissipated at the single transducer. One or more binding events in the thin structure alter these resonances, allowing the binding events to be detected. As described below, other electronics and/or circuitry may similarly be utilized in an embodiment employing a SAW sensor having only one interdigital transducer.

One embodiment of the invention relates to the microsensors, wherein at least two adjacent IDTEs have a height from 10 nm to 1 micron and the micro channel between said adjacent IDTE has a width from 100 nm to 10 microns.

One embodiment of the invention relates to the microsensors, wherein at least two adjacent reflectors have a height from 10 nm to 1 micron and the micro channel between said adjacent reflectors has a width from 100 nm to 10 microns.

One embodiment of the invention relates to the microsensors, wherein at least two adjacent I DTE/reflectors junctions have a height from 10 nm to 1 micron and the micro channel between said adjacent structures has a width from 100 nm to 10 microns.

One embodiment of the invention relates to the microsensors, wherein the molecular recognition component is selected from the group consisting of nucleic acids, nucleotide, nucleoside, nucleic acids analogues such as PNA and LNA molecules, proteins, peptides, antibodies including IgA, IgG, IgM, IgE, enzymes, enzymes cofactors, enzyme substrates, enzymes inhibitors, receptors, ligands, kinases, Protein A, Poly U, Poly A, Poly lysine, triazine dye, boronic acid, thiol, heparin, polysaccharides, coomassie blue, azure A, metal-binding peptides, sugar, carbohydrate, chelating agents, prokaryotic cells and eukaryotic cells.

One embodiment of the invention relates to the microsensors, wherein the molecular recognition component immobilized on the units of a set of SAW resonator is not the same. Said recognition component may be different, but binds to different parts of the same analyte or it may bind to different analytes.

One embodiment of the invention relates to the microsensors, wherein the sample is selected from the group consisting of blood, serum, plasma, ascites, faeces, spinal core fluids, urine, smears and saliva. One embodiment of the invention relates to the microsensors, wherein the SAW device is a SAW filter unit type.

One embodiment of the invention relates to a handheld device for detecting target ana- lyte in a sample wherein said device comprises the microsensors.

One embodiment of the invention relates to the use of the microsensors for measuring a signal upon detection of a target analyte in a sample.

Target analyte may be any molecule such as biological molecules such as e.g. nucleic acids, proteins, peptides, antibodies, enzymes, carbohydrates, chemical compounds, and gasses. Other target analyte may be selected from the group consisting of Troponin 1 , Troponin T, BNP, H-FABP, allergens or immunoglobulins such as IgE. In certain embodiments, the target analyte is capable of binding more than one molecular recognition component.

One embodiment of the invention relates to the use, wherein the target analyte is selected from the group consisting of Troponin I, Troponin T, BNP, an H-FABP, an allergen and IgE.

One embodiment of the invention relates to a microsensor for measuring a signal upon detection of a target analyte in a sample, said microsensor comprising at least one set of two or more surface acoustic wave (SAW) resonator units, each unit comprising: (a) a piezoelectric substrate, (b) at least one interdigital transducer electrode (IDTE) structure having three- dimensional micro channels formed within the IDTE structure,

(c) at least one reflector (REF) structure having three-dimensional micro channels formed within the reflector structure, and

(d) optionally, a molecular recognition component (MRC), wherein at least one molecular recognition component (MRC) (d) is immobilized in the micro channels of the IDTE structure of at least one of the units, and wherein at least one molecular recognition component (MRC) (d) is immobilized in the micro channels of the reflector (REF) structure of at least one of the units, and wherein the set of SAW resonator units comprises at least two units with differential immobilization of the molecular recognition component (MRC) (d), which units with differential immobilization are selected from the group of (a) a unit with IDTE and REF structures having micro channels with immobilized MCR, (b) a unit with IDTE and REF structures having micro channels without immobilized MCR₁

(c) a unit with IDTE structure having micro channels with immobilized MCR and REF structure having micro channels without immobilized MCR, and (d) a unit with REF structure having micro channels with immobilized MCR and IDTE structure having micro channels without immobilized MCR.

The present invention especially relates to (1) a microsensor for detecting the presence of a target analyte in a test sample solution comprising; at least one surface acoustic wave (SAW) resonator units comprising; a piezoelectric substrate; a plurality of inter- digital transducer electrode (IDTE) structures and reflector structures on a surface of said substrate; wherein three-dimensional micro channels are formed between said IDTE and reflector structures; having at least one molecular recognition component differentially immobilized in said three-dimensional micro channels formed between said IDTE structures or reflector structures; wherein the word differentially immobilized relates to at least one molecular recognition component immobilization on one homogeneous set of either IDTE structures or reflector structures but not on both sets of structures; having an identical set of homogeneous reference IDTE or reflector structures without a molecular recognition component immobilized; having the target analyte in the test sample binding the differentially immobilized molecular recognition component.

Further embodiments of the present invention are:

A microsensor (1) as the above, wherein a signal change of frequency, phase or am- plitude occurs due to the molecular recognition component binding the analyte in the test sample. Said signal can either be of decreasing or increasing nature, depending on which homogeneous structures have been modified.

The microsensor as the above (1 ), wherein the amplitude of at least one SAW resona- tor unit is adjusted for optimal conditions for at least one molecular recognition component to react with the target analyte in the test sample.

The microsensor as the above (1) comprising said homogeneous reference structures, wherein said homogeneous reference structures do not have any molecular recognition component immobilized; wherein said homogeneous reference structures are capable of subtracting the signal of said homogeneous reference structures from the said signal of identical signal homogeneous structures having the molecular recognition compo- nent immobilized; wherein the delta signal can be directly correlated to the concentration of the target analyte in the test sample.

The microsensor as the above (1 ), wherein both reference structures and structures with immobilized molecular recognition components are present on the same piezoelectric substrate.

The microsensor as the above (1 ), wherein sets of IDTE structures with immobilized molecular recognition components are present with reference reflector structures on the same piezoelectric substrate; having a second piezoelectric substrate, where sets of reflector structures with immobilized molecular recognition components are present with reference IDTE structures; wherein the delta signal between reference structures and immobilized structures can be directly correlated to the concentration of the target analyte in the test sample.

The microsensor as the above (1 ), having at least one differential coating layer localized on the said homogeneous structures before having the molecular recognition component immobilized.

The microsensor as the above (1), wherein the coating layer consists of or comprises, without being limited to, one ore more materials selected from gold (Au), silver (Ag), SiO2, aluminium (Al) or any kind of polymer material.

The microsensor as the above (1 ), wherein a signal change of frequency or phase oc- curs due to a liquid/solid volume ratio change in said three-dimensional micro channels between either IDTE structures or between reflector structures or channels between both structures.

The microsensor as the above (1 ), wherein at least two adjacent IDTEs have a height from 10 nm to 1 micron and the micro channel between said adjacent electrodes has a width from 100 nm to 10 microns.

The microsensor as the above (1 ), wherein at least two adjacent reflectors have a height from 10 nm to 1 micron and the micro channel between said adjacent electrodes has a width from 100 nm to 10 microns. The microsensor as the above (1), wherein at least two adjacent I DTE/reflectors junctions have a height from 10 nm to 1 micron and the micro channel between said adjacent structures has a width from 100 nm to 10 microns.

The microsensor as the above (1 ), wherein at least one insulation coating on the IDTE and reflector structures consists of, but is not limited to, titanium, SiO2, a dielectric thin film, quartz or any kind of polymer material.

The microsensor as the above (1), wherein the piezoelectric substrate consists of, but is not limited to, LiTaO.sub.3 and quartz.

The microsensor as the above (1), wherein the molecular recognition component is selected from the group consisting of nucleic acids, nucleotide, nucleoside, nucleic acids analogues such as PNA and LNA molecules, proteins, peptides, antibodies including IgA, IgG, IgM, IgE, enzymes, enzymes cofactors, enzyme substrates, enzymes inhibitors, membrane receptors, kinases, Protein A, Poly U, Poly A, Poly lysine, triazine dye, boronic acid, thiol, heparin, membrane receptors, polysaccharides, coomassie blue, azure A, metal-binding peptides, sugar, carbohydrate, chelating agents, prokaryotic cells and eukaryotic ceils.

A microsensor as the above (1), wherein the test sample solution is selected from the group consisting of blood, serum, plasma, ascites, faeces, spinal core fluids, urine, smears and saliva.

A handheld device for detecting target analytes comprising the microsensor according to the invention.

A microsensor for detecting the presence of a target analyte in a test sample solution comprising; at least two surface acoustic wave (SAW) resonator units each comprising: a piezoelectric substrate; at least one IDTE and at least two reflectors disposed on each side of said IDT electrodes; first said SAW resonator unit having a molecular recognition component immobilized on top and in between the three-dimensional micro channels formed between said electrodes structures and on top and in between the three-dimensional micro channels formed between said reflectors structures; wherein second said SAW resonator unit does not have any molecular recognition component immobilized. Preferably, the SAW device in the microsensor according to the invention is a SAW device of the SAW filter unit type.

In one preferred embodiment the target analyte is Troponin I or Troponin T.

In one preferred embodiment the target analyte is BNP.

In one preferred embodiment the target analyte is an H-FABP.

In one preferred embodiment the target analyte is an allergen or IgE.

The present invention may be used in combination with the invention disclosed in the application "Bio surface acoustic wave (SAW) resonator amplification for detection of a target analyte".

Claims

1. A surface acoustic wave (SAW) resonator unit comprising:

(a) a piezoelectric substrate, (b) at least one interdigital transducer electrode (IDTE) structure,

(c) at least one reflector structure, and

2. The resonator unit according to claim 1 , wherein the at least one molecular recognition component is immobilized only on the IDTE structure(s).

3. The resonator unit according to claim 2, wherein the at least one molecular recognition component is immobilized both in the micro channels as well as on top of the IDTE structure(s).

4. The resonator unit according to claim 1 or 2, in which the at least one molecular recognition component is immobilized only, or substantially only, in the micro channels of the IDTE structure(s).

5. The resonator unit according to any of claims 1-4, wherein the molecular recognition component is immobilized in an immobilization matrix.

6. The resonator unit according to claim 5, wherein the matrix comprising the molecu- lar recognition component changes viscosity in response to binding between the analyte and the molecular recognition component.

7. The resonator unit according to any of claims 1-6, wherein at least two adjacent IDTEs have a height from 10 nm to 1 micron and the micro channel between said adjacent IDTEs has a width from 100 nm to 10 microns.

8. The resonator unit according to any of claims 1-7, wherein at least two adjacent reflectors have a height from 10 nm to 1 micron and the micro channel between said adjacent reflectors has a width from 100 nm to 10 microns.

9. The resonator unit according to any of claims 1-8, wherein at least two adjacent I DTE/reflectors junctions have a height from 10 nm to 1 micron and the micro channel between said adjacent structures has a width from 100 nm to 10 microns.

10. The resonator unit according to any of claims 1-9, wherein the molecular recogni- tion component is selected from the group consisting of nucleic acids, nucleotide, nucleoside, nucleic acids analogues such as PNA and LNA molecules, proteins, peptides, antibodies including IgA, IgG, IgM, IgE, enzymes, enzymes cofactors, enzyme substrates, enzymes inhibitors, receptors, ligands, kinases, Protein A, Poly U, Poly A, Poly lysine, triazine dye, boronic acid, thiol, heparin, polysaccharides, coomassie blue, azure A, metal-binding peptides, sugar, carbohydrate, chelating agents, prokaryotic cells and eukaryotic cells.

11. A microsensor comprising at least one surface acoustic wave (SAW) resonator unit according to any of claims 1-10.

12. The microsensor according to claim 11 comprising at least one set of surface acoustic wave (SAW) resonator units according to any of claims 1-10.

13. The microsensor according to claim 11 or 12 further comprising at least one refer- ence surface acoustic wave (SAW) resonator unit that does not comprise immobilized molecular recognition components.

14. The microsensor according to claim 13 comprising at least one set of reference surface acoustic wave (SAW) resonator units that do not comprise immobilized mo- lecular recognition components.

15. The microsensor according to any of claims 11-14, wherein the at least one molecular recognition component is immobilized only on the IDTE structure of the first unit and on the reflector of the second unit.

16. The microsensor according to claims 11-15, wherein the units of a set are placed on the same piezoelectric substrate.

17. The microsensor according to claims 11-15, wherein the units of a set are placed on separate piezoelectric substrates.

18. A handheld device for detecting target analytes comprising the microsensor of any of claims 11-17.

19. The microsensor according to claims 11-18, wherein the at least one molecular recognition component is immobilized only, or substantially only, in the micro channels of the IDTE and/or the reflector structures.

20. The microsensor according to claims 11-19, wherein the molecular recognition component immobilized on the two units of a pair is not the same.

21. The microsensor according to claims 11-20, wherein the sample is selected from the group consisting of blood, serum, plasma, faeces, spinal core fluids, urine, smears and saliva.

22. Use of the microsensor according to claims 11-20 for measuring a signal upon de- tection of a target analyte in a sample.

23. Use according to claim 22, wherein the target analyte is selected from the group consisting of Troponin I, Troponin T, BNP, an H-FABP, an allergen and IgE.