Method and Apparatus for Detecting a Substance of Interest

Field of the invention

The present invention relates to a sensor for use in detecting a substance of interest. In particular, it relates to a sensor in which the presence of a substance of interest is detected by monitoring a change in viscoelastic response of the sensor upon binding of the substance to the sensor. The invention also relates to the preparation of such a sensor and to its use in detecting substances of interest.

Background to the invention

The development of assays capable of detecting multiple chemical or biological analytes is of considerable interest in a number of fields including civilian and military defence, medical diagnostics, process monitoring in the chemical and bioprocessing industries, environmental monitoring and pharmaceutical screening.

Biological and chemical assays for multiple analytes are known in the art. Methods which have been described previously include the use of an array of surface- immobilised receptors for analytes of interest, where the position of the receptor in the array identifies the analyte, with detection of binding by, for example, fluorescence. Another method employs microspheres, identified by a fluorescent 'barcode', to support immobilised receptors. Both of these types of assay rely on the analyte, or a binding agent that is competitive with the analyte or a molecule that can subsequently bind to the analyte, being labelled with a fluorescent or radioactive marker for detection. The use of labels is disadvantageous, however, as the need to synthesise labelled molecules precludes a high throughput assay of drug libraries, for example. Moreover, in cases where a labelled reporting molecule cannot be produced, no assay can be performed.

Label-free methods have been described, including assays that rely on techniques such as mass spectrometry or surface plasmon resonance. Although such methods can work well, they suffer from the disadvantages that they generally require expensive

infrastructure, may be difficult to operate in a high throughput manner and the equipment used is not readily portable, limiting the potential applications.

Alternative label free assays using a flexible cantilever which is deflected upon binding of an analyte to its surface are described, for example in EP 1226437B to IBM. This approach shows promise but the devices are fragile and multiplexed versions with many cantilevers on one chip are expensive and highly sensitive to vibration, so that portable versions are difficult to realise.

The use of functionalised polymers to detect binding of analytes has also been proposed. Functionalised polymers such as hydrogels that are sensitive to pHhave been described by Siegel et. aL, (Macromolecules 21, 3254-3259 (1988)) and Verestiuc et. al. (International Journal of Pharmaceutics 269, 185-194, (2004)) and temperature sensitive hydrogels have been described by Chen et. al. (Nature 373, 49±52 (1995), Yoshida, et al. Nature 374, 240±242 (1995)). Such materials are often referred to as 'stimuli- responsive hydrogels' or 'smart hydrogels'. In some cases the binding of an analyte to the material results in a swelling which can be detected. This swelling response requires relatively high concentrations of the analyte to generate a measurable size change. Examples which have been reported include a hydrogel that has a sol-gel transition sensitive to the concentration of glucose (Lee, S. J., Park, K., Journal of Molecular Recognition, Vol. 9, 549-557 (1996)). Miyata et. al, Nature, 399, 766-769, (1999) describe a hydrogel material, which swells reversibly in solution in response to a specific antigen. In this case, the hydrogel was prepared by grafting an antigen and corresponding antibody to the polymer network, so that binding between the two introduces crosslinks in the network. Competitive binding of free antigen triggers a change in gel volume owing to breaking of these non-covalent crosslinks. To date, the majority of applications for smart hydrogels which have been proposed have been in the fields of drug delivery and biomimetic tissues.

The use of a functionalised hydrogel to detect glucose, thyroxin or oestrogen is proposed in GB2383846A (Sentec). In the embodiment described therein, a millimetre-sized mass of gel that specifically binds the analyte encapsulates a piece of magnetostrictive material whose resonance, when driven by an external magnetic field, is affected by the

change in mechanical properties of the hydrogel when the analyte binds. This device is designed to be placed inside a person, for example subcutaneously, and the sensor interrogated without the need for wires by a magnetic device strapped to the skin. Such a device could not be used effectively to detect more than one analyte in a multiplexed assay because there would be likely to be insurmountable interference between magnetostrictive elements in such an array. Moreover, such as array would be unmanageably large because of the size of the magnetostrictive material required to obtain a good signal to noise ratio in the measurement.

There remains a continuing need for the development of improved sensors for the detection and measurement of analytes within chemical or biological systems and in particular for the development of sensors which can be used to detect multiple analytes in a multiplexed assay.

Summary of the invention

The present inventors have found that analyte binding can be detected with improved sensitivity by monitoring the change hi viscoelastic response of a polymer material capable of binding the analyte upon binding of the analyte, affording the possibility of improved sensors which are capable of detecting multiple analytes without the need for the analytes or any other molecules to be labelled.

According to a first aspect, therefore, the present invention provides a sensor system for detecting a substance of interest comprising:

(a) a viscoelastic material capable of interacting with the substance of interest, the material exhibiting a change in viscoelastic response upon interaction with said substance; and

(b) means for detecting a change in the viscoelastic response of the viscoelastic material when the viscoelastic material interacts with the substance of interest.

In a further aspect, the invention also provides an array comprising a plurality of such sensors, wherein each of the plurality of sensors comprises a viscoelastic material

capable of interacting with a substance of interest, the substance with which each sensor is capable of interacting being the same or different.

The invention also provides a method for detecting one or more substances of interest in a sample using a sensor or array of sensors according to the above aspects of the invention, the method comprising the steps of:

(a) exposing the sample to the sensor or array of sensors;

(b) determining whether the sample includes the substance of interest by detecting a change in viscoelastic response of the viscoelastic material of the sensor upon interaction with the substance of interest .

Also provided is a method for measuring the concentration of a substance of interest in a sample comprising the steps of :

(a) detecting the presence of the substance of interest according to the above method; (b) measuring the change in viscoelastic response upon interaction of the substance of interest with the sensor; and

(c) determining from the measured change in viscoelastic response the concentration of the substance of interest.

In a further aspect, the invention also provides a viscoelastic material having embedded therein one or more particles capable of being driven into oscillation by an applied external force and an array comprising a plurality of such materials.

It will be appreciated that any one or more of the above aspects of the invention may be combined in any combination.

By means of the invention, label free sensors which provide a quantitative measure of the viscoelasticity or rheology of the sensor material and so exhibit greater sensitivity to analyte concentration are provided. The monitoring method allows for a high degree of miniaturization and enabling multiple analytes to be assayed in small volumes of sample. This represents a significant advantage over methods such as that described in GB 2383846A, which does not lend itself to use in multianalyte arrays or with small volumes for the reasons discussed above.

Detailed description of the invention

By 'viscoelastic material' is meant a material which exhibits both viscous and elastic responses when subjected to stress or strain. A viscoelastic liquid will deform and flow under the influence of an applied shear stress, for example, but will slowly recover from some of the deformation when the stress is removed.

As used herein, by 'capable of interacting with the substance of interest' is meant that the viscoelastic material binds the substance of interest, either directly to the material or indirectly through interaction with one or more intermediate molecules capable of linking the substance of interest to the material.

By 'viscoelastic response' of the material is meant the elastic and dissipative response of the material when subjected to shear or other stress.

The viscoelastic material for use according to the invention may be any material which shows binding specificity for the substance of interest and which exhibits a change in viscoelastic response upon interaction with the substance.

Suitably, the viscoelastic material for use according to the invention may be a polymer, biopolymer or hydrogel formed from a polymer, a low molecular weight organic material or a clay with appropriate salts in water.

Preferably, the viscoelastic material is a polymer material which is permeable to the solution of the substance of interest and is in the form of a three dimensional network of polymer chains, a gel or particularly preferably a hydrogel.

Typically the polymer may be selected from agarose, polyacrylamide, polyurethane, poly(n-isopropylacrylamide), vinylpyrrolidinone, bis(trimethoxysilyethyl)benzene.

Conveniently, the viscoelastic material for use according to the invention may be functionalised by techniques conventional in the art using an appropriate linking molecule capable of linking the substance of interest to the material. Suitably the

material is chemically modified with a receptor such as an antibody having binding specificity for the substance of interest.

It will be appreciated that the choice of viscoelastic material, the density of the polymer network, the nature of the linking molecule and the number of linking molecules per unit volume may all be varied to optimize the sensitivity and specificity of the sensor system according to the invention.

The sensitivity and specificity of the sensor system for a given substance of interest may be improved by tuning the pore size of the polymer network, or at least part thereof, to the molecular size of the substance of interest. Sensitivity to small analytes may suitably be improved, for example, by controlling the pore size of the material such that larger analytes are prevented from entering the polymer network. Alternatively, a composite material , for example, having layers of gel with different pore sizes and functionalized to have different binding specificities may be employed, enabling analytes of varying molecular size to be detected.

In one embodiment, the sensor system comprises a viscoelastic material which is maintained close to a phase transition, such as a sol-gel transition, by appropriate choice of temperature and pH conditions. Binding of the analyte substance induces the transition, causing a major change in viscoelastic response and hence improving the sensitivity of the sensor.

The method of the invention is applicable to any analyte substance of interest. Suitably, for example, the substance of interest may be selected from virus particles, bacteria, proteins, antibodies, antigens, nucleic acids, peptides, small molecules such as toxins, drugs of abuse, explosives or metabolites and the method of the invention may find application in the fields of medical diagnostics, civilian and military defence, process monitoring or pharmaceutical screening.

In use, according to the invention, the sensor system is conveniently exposed to a solution of the sample to be analysed. If the sensor material has binding specificity for the substance of interest, for example if it has been functionalized with an appropriate

receptor for the substance of interest, then binding of the analyte substance of interest to the material can occur, resulting in a change in the viscoelastic properties of the material. This change in viscoelastic properties may be caused simply by the presence of the analyte in the voids between the polymer molecules or by crosslinking the polymer molecules.

The change in viscoelastic response of the viscoelastic material upon binding of the analyte of interest can be determined by a variety of techniques.

These include recording the thermally driven Brownian motion of the system and analyzing the results by applying one of the appropriate well established theoretical models of viscoelasticity. Other methods which may be used include optical method such as dynamic light scattering or diffusing wave spectroscopy , or touching or embedding a physical probe (e.g. an atomic force microscope tip or similar) within the polymer material and recording the response of this mechanical system.

Suitably, the means for determining the change in viscoelastic response upon interaction with the substance of interest comprise means for driving the viscoelastic material into motion and means for monitoring the motion of the viscoelastic material in response to the driving force, the motion in response to the driving force depending on changes in the viscoelastic properties of the material which in turn are dependent on changes in concentration of the analyte substance of interest.

Conveniently, a method of measuring the viscoelastic response may be used in which the viscoelastic material is driven into oscillation by an external device (for example, a piezo ceramic actuator or acoustic actuator) or using a magnetic or electric field if the material is responsive to magnetic or electric fields, and the amplitude and phase of the response of the material to this driving force is monitored . The response may suitably be monitored by tracking a particle within the material, or tracking the strands of the material themselves, or tracking the position of a datum on or within the viscoelastic material , the tracking conveniently being performed using an optical imaging system (such as a CCD camera or photodiode or the like) or by reflecting or scattering a laser or other light source from the object and monitoring the reflected or scattered light using a

position sensitive detector (such as split photodiode). The motion of the system is recorded, analysed and well established theoretical models of viscoelasticity can then be applied to the data to determine the elastic and dissipative response of the material.

Preferably, the viscoelastic response may be measured by a method in which one or more particles, embedded within the viscoelastic material, or other datum within or upon the material are driven into oscillation by an applied external force and the amplitude and phase of the response of the particles or datum to this driving force is monitored. By 'datum' is meant an identifiable feature whose position can be monitored. This may be a marker of some kind which is placed on or within the material or an inherent but readily monitorable feature of the material itself. Depending on the properties of the particle, the particle can be driven into oscillation using either an electric or magnetic or acoustic field, for example, and the position of the particle monitored using optical microscopy methods (for example using an optical imaging system e.g. CCD camera or photodiode or similar) or by reflecting or scattering a laser or other light source from the object and monitoring the reflected or scattered light using a position sensitive detector e.g. a split photodiode. Preferably, the particles embedded within the viscoelastic material are capable of being driven into oscillation by an applied magnetic field. The motion of the particle can be analysed by applying any appropriate, well established theoretical model of viscoelasticity and the elastic and dissipative response of the material determined accordingly.

Depending on the choice of particle size, either molecular or bulk viscoelastic responses of the viscoelastic material may be determined by this method. If the particle embedded within the material is smaller than the pore size of the network of the material, for example, it contacts single or only a few of the material strands such that measurement of the movement of the particle gives a measure of the molecular response of the viscoelastic material. If the particle is larger than the pore size of the viscoelastic material network , however, then it contacts many strands of the material and so its motion measures a bulk response of the material.

Determining the viscoelastic response of the sensor material upon binding of the substance of interest provides a much more sensitive measure of the change in the

sensor on binding of the analyte substance, and hence greater sensitivity to analyte concentration, than methods described hitherto such as the method described in GB 2383846A.

The method of GB 2383846A employs a magnetostrictive wire which means that its dimensions (but not its position) change in response to the variation of an applied magnetic field. The applied magnetic field must be turned off to detect the response of the wire, the method depending on detecting the magnetic field re-emitted when the wire relaxes which itself may be dependent on, for example, the viscosity of the medium. This method is not concerned with measuring the motion of the wire and so does not provide a quantitative measure of the viscous and elastic response of the polymer material. In contrast, the method of the present invention provides a quantitative measure of the complex linear or nonlinear viscoleastic response of the viscoelastic material and so provides a far more sensitive measure of the change in the sensing material upon binding of the analyte substance of interest.

Detection of an analyte by means of the light and acoustic energy propagational properties on an analyte-responsive polymer is disclosed in WO 94/02852. This describes the use of a use of a thin cross linked polymer film attached to the surface of a quartz crystal microbalance to detect the presence of selected analytes and demonstrates its use in sensing pH..The change in propagational properties is tentatively attributed inter alia to changes in viscoelasticity of the analyte-responsive polymer but the experimental data presented only shows sensitivity to the pH environment of the polymer, suggesting that this sensor only responds to the general chemical environment of the polymer rather than allowing molecule specific detection of analytes as in the present invention. The response of the sensor described in WO 94/02852 is driven by changes in electrostatic forces in the polymer sensor element and is less specific and more environmental than the method of the present invention. Moreover, quartz crystal microbalances are not well suited to use in multiplexed assays capable of detecting multiple analytes simultaneously.

A scheme presented by Cheeke et al (1996 IEEE Ultrasonics Symposium, page 449-452) uses a surface acoustic wave sensor to measure the changes in visco-elastic properties of a polymer in response to changes in ambient humidity. This system again does not posses a truly molecule specific response and is poorly suited to the fabrication of multiplexed sensors capable of detecting multiple analytes simultaneously

A multiplexed sensor device according to the invention which is sensitive to more than one different analyte substance simultaneously may conveniently be prepared by placing a plurality of sensors, each sensitive to a different analyte substance, on a solid support such as silicon, metal, plastic or glass in a patterned array of spots The position of the spot within the array is indicative of the receptor and therefore the specificity to analyte of that gel spot. The gels spots may be placed on the solid support by robotic spotting, ink jet printing or contact printing for example and may be of any suitable shape or size. Preferably the gel spots are of micron dimensions such that an array with overall dimensions of tens to hundreds of microns can be fabricated, enabling the entire array to be exposed to the analytes in the solution using only small sample volumes.

The invention may be further illustrated by way of example only with reference to the following figures in which:-

Figure IA shows a polymer material for use according to the invention.

Figure IB shows the polymer material of Figure IA with analyte bound to the polymer network

Figure 1C shows an array of sensors according to the invention

Figures 2 A and 2B show an embodiment of a sensor system according to the invention.

Referring to Figure IA, this shows a polymer network (1) containing many voids that can be filled with solvent. This material is chemically functionalised with receptors (2) capable of binding an analyte of interest.

In Figure IB there is shown a polymer material (as in Figure IA) which has been exposed to a solution of an analyte (3), the analyte binding to the polymer network (4).

The same or several different polymer materials each functionalised with a different receptor for a different analyte can be spotted onto a solid support to create an array

sensor that is sensitive for multiple analytes as is shown in Figure 1C. Preferably the polymer spots are of micron dimensions so that small volumes (hundreds of microlitres or less) of sample can be used to expose the entire array simultaneously.

Figure 2A shows magnetic particles (7) incorporated in the polymer material which can be driven back and forth by a magnetic field gradient, the movement of the particles depending on the viscoelastic properties of the polymer network, a change in viscoelastic response being observed if an analyte has bound to the polymer. The magnetic particles may be ferro- or paramagnetic.

The magnetic field gradient required to drive the magnetic particles can be provided by a solenoid or electromagnetic (8) or other similar device placed in proximity to the sensor material as shown in Figure 2B. The position of the magnetic particles is recorded optically either by using scattered light or fluorescence and a video camera or other suitable optical detector (not shown). Control electronics, optics, a light source, a detector and data recorder may also be provided.