Sensing device for obtaining rotational properties of a rotatable object in a fluid

Abstract

A sensing device for sensing a rotational property and / or a further property associated with one or more objects, for example, nanometre or micron sized objects in a fluid, the device comprising: two or more driving elements and associated driving circuitry configured to drive said driving elements using two or more respective signals characterised by a difference in at least their phase and / or other electrical property; at least one sensing element configured to sense a response signal in response to driving the driving elements using the two or more signals, wherein a difference in at least the phase and / or other electrical property of the two or more respective signals causes the sensed response signal to be dependent on at least a difference in impedance between each of the two or more driving elements and the at least one sensing element such that a rotational property and / or at least one further property associated with the one or more objects is obtainable from the sensed response signal, wherein the rotational property and / or at least one further property comprises a speed of rotation and / or a frequency of rotation and / or a direction of rotation and / or a change in direction of rotation.

Description

[0001] SENSING DEVICE FOR OBTAINING ROTATIONAL PROPERTIESOF A ROTATABLE OBJECT IN A FLUID

[0002] Field

[0003] The present invention relates to a sensing device and method, for example, a sensing device for obtaining rotational properties and / or further properties associated with one or more rotatable objects in a fluid.

[0004] Background

[0005] The bacterial flagellar motor (BFM) is a rotary molecular machine whose evolutionary function is to enable the propulsion of a bacterium by rotating the flagellum. The motor may serve as part of a multimodal sensor. For example, the motor may be utilized as part of a non-invasive single cell voltmeter (where it measures the electrochemical gradient of the cell itself), a mechanosensor for a viscous load or flow detection, and a chemosensor allowing detection of specific molecules.

[0006] The response time of known BFM based sensors may be short and sensitivity may be such that nanoMolar (nM) concentrations may be detected. In some sensors, the relevant output for a given sensing modality is the rotational speed or direction of the motor. Known available techniques to measure individual motor speed and direction, while high in spatiotemporal resolution, may be low in throughput. Known sensors may also be complex to build and rely on optical detection.

[0007] Summary

[0008] In a first aspect there is provided a sensing device for sensing a rotational property and / or a further property associated with one or more objects, for example, nanometre or micron sized objects, the device comprising: two or more driving elements and associated driving circuitry configured to drive said driving elements using two or more respective signals characterised by a difference in at least their phase and / or other electrical property; at least one sensing element configured to sense a response signal in response to driving the driving elements using the two or more signals, wherein a difference in at least the phase and / or other electrical property of the two or more respective signals causes the sensed response signal to be dependent on at least a difference in impedance between each of the two or more driving elements and the at least one sensing element such that a rotational property and / or at least one further property associated with the one or more objects is obtainable from the sensed response signal. The rotational property and / or at least one further property may comprise a speed of rotation and / or a frequency of rotation and / or a direction of rotation and / or a change in direction of rotation. The one or more object may be in a fluid.

[0009] The sensed response signal is dependent on at least a frequency of rotation of the object and a driving signal frequency. The sensed response signal may comprise a modulated signal dependent on the driving signal frequency and a frequency of rotation. The rotational property and / or at least one further property may be obtainable by demodulating the sensed response signal using the driving signal frequency.

[0010] The object may comprise and / or be attached to a bacterial flagellum. The object may be attached to a further object, and the rotational property and / or at least one further property may be a property of the further object. The rotation or movement of the object may be due to the rotary motor of the bacteria flagellum. The rotation of the object may be due to the action of a rotating motor. The object may comprise a bead or other detectable marker attached to a bacteria flagellum.

[0011] The object may rotate and / or revolve and / or exhibit rotational and / or substantially circular movement and / or substantially elliptical movement. The object may move about an axis of rotation thus causing said rotational and / or circular and / or elliptical movement about said axis. The speed of rotation and / or frequency of rotation may comprise the speed and / or frequency of moving around axis. The direction of rotation may comprise the direction of movement about said axis.

[0012] The one or more objects may move in a closed trajectory and / or orbit. The closed trajectory and / or orbit may be about an axis and / or a point. The speed of rotation may comprise a speed of traversing the closed trajectory and / or orbit. The frequency of rotation may comprise a frequency of traversing the closed trajectory and / or orbit. The direction of rotation may comprise a direction of traversing the closed trajectory and / or orbit.

[0013] The one or more objects may rotate about a point or axis defined separately from the object. The one or more objects may comprise an object attached or otherwise secured to a rotating object. The rotating object may be rotating about a body axis. Rotation of the rotating body may cause the object to move in a circular and / or elliptical motion about said body axis. The device may be configured to obtain a speed and / or frequency of rotation and / or direction of rotation of an object attached to a bacteria flagellum. The device may be configured to measure a property of object attached the bacteria flagellum or the bacteria flagellum.

[0014] The sensing device may comprise a sensing surface for contacting the fluid. The two or more driving elements and / or at least one sensing element may be arranged at and / or disposed in said sensing surface.

[0015] The object may be attached to a bacteria flagellum and the object may rotate and / or exhibits rotational and / or substantially circular or elliptical motion due to the rotary motor of the bacteria flagellum.

[0016] The sensing device may comprise an attachment and / or tethering means on a sensing surface or on a further surface for attaching or tethering at least part of the one or more objects or a further object attached to the object.

[0017] The attachment or tethering means may position the object substantially between a driving element and a sensing element. The attachment or tethering means may position the object at a position substantially above the at least one sensing element. The attachment or tethering means may comprise polymer patterns. The attachment or tethering means may comprise positively charged polymers stamped onto a sensing surface and / or further surface using PDMS stamps and / or other method, for example, microprinting. The attachment or tethering means may comprise polymer patterning along the sensing surface and / or a further surface.

[0018] The attachment or tethering means may be such that the object rotates on a trajectory such that, at least part of the trajectory overlaps with a region between at least one of the driving elements and the at least one sensing element.

[0019] The attachment or tethering means may be configured to position the object at or near to a surface area of the driving and / or sensing elements. The attachment and / or tethering means may substantially position the object at a position at or around the at least one sensing element.

[0020] The attachment means may comprise attaching means. The attachment and / or tethering means may comprise an attacher or tetherer. The attachment and / or tethering means may comprise a bead or other marker assay

[0021] The attaching and / or tethering means may be configured to attach and / or tether the object thereby to substantially prevent linear movement of said object and / or substantially fix a rotational axis of said object and / or to permit rotation and / or circular or elliptical movement of said object.

[0022] A first sensing region may be defined between the at least one sensing element and the first driving element and a second sensing region may be defined between the at least one sensing element and the second driving element. The attachment or tethering means may attach and / or tether the object such that the object follows a path in at least part of both the first and second sensing regions.

[0023] The path may comprise a closed trajectory. The path may comprise an orbit. The one or more rotational and / or further properties may relate to movement of the further object on said path.

[0024] The attachment and / or tethering means may comprise one or more surfaces comprising one or more attaching and / or tethering sites operable to attach and / or tether said object or a further object secured to said object.

[0025] The sensing device may comprise an attachment or tethering means on the sensing surface and / or on a further surface for attaching or tethering at least part of the one or more objects or a further object attached to the object thereby to substantially prevent linear movement and / or substantially fix a rotational axis of the object and / or permit rotation of said object. The further surface and the sensing surface may from part of a channel. The further surface may be an upper or opposing or other surface, relative to the sensing surface. The two or more signals may be characterised by a difference in at least their phase and / or other electrical property such that they at least partially cancel. The two or more signals may be characterised in their phase and / or other electrical property such that the electric field produced by the two or more signals at least partially cancel.

[0026] The object may have at least one dimension in the range 1 nanometre (nm) to 100 microns (pm), optionally, between 10 nm and 10 microns.

[0027] The device may comprise an integrated circuit and wherein the two or more driving elements and the at least one sensing element comprises electrodes integrated into the integrated circuit. The sensing device may comprise or may be a sensor.

[0028] The two or more driving elements comprise a first driving element and a second driving element and wherein the difference in impedance comprises a difference between a first impedance between the first driving element and the at least one sensing element and a second impedance between the second driving element and the at least one sensing element.

[0029] The sensed response may comprise a combination of sensed responses from each of the two or more signals, wherein the difference in their phase and / or other electrical property of the two or more signals is such that the sensed responses substantially cancel to allow a contribution from the rotational motion of the objects in said fluid to be obtainable from the sensed response signal. The sensed response may comprise a combination of a first response from the first signal and a second response from the second signal. The sensed response may comprise a modulated signal.

[0030] The device may comprise processing circuitry configured to process the sensed electrical response to determine the rotational property and / or at least further property associated with the object.

[0031] The processing circuitry may comprise signal processing circuitry. The processing circuitry may comprise a demodulation module for demodulating the sensed response, signal. The rotational property and / or at least one further property may comprise a rotational property of the object or an attached rotary motor or a further property of the fluid based on the rotational property of the object or attached rotary motor. Obtaining the rotational property may comprise obtaining a derivative of the sensed response signal and identifying one or more discontinuities in the derivate. The one or more discontinuities may indicate or represent a change in direction of rotation.

[0032] The two or more respective signals may comprise driving signals. The two or more respective signals may have a signal frequency and / or a range of signal frequencies and wherein the sensed response signal is dependent on the signal frequency. The driving signals may be produced at a signal frequency and / or a range of signal frequencies and wherein the sensed response is dependent on the signal frequency.

[0033] The driving circuitry may be configured to produce a pair of differential and / or complimentary signals.

[0034] The two or more respective signals, optionally a pair of the two or more signals, have a phase difference between 135 and 180 degrees, optionally wherein the pair of signals are substantially out of phase. The two or more respective signals may be a pair of signals that are out of phase or have a phase difference between 135 and 180 degrees.

[0035] The driving circuitry may be configured to drive the two or more driving elements at a signal frequency in the range of 100 kHz to 100 MHz frequency signal. The two or more respective signals may comprise oscillating and / or alternating current signals.

[0036] The two or more respective signals may drive said driving elements to produce electrical fields. The produced electrical fields may comprise at least a phase difference and / or a difference in a further property.

[0037] The rotational property may comprise a rotational speed and / or frequency of rotation of the object. The rotational property may comprise a rotational direction. The rotation property may comprise a change in rotational direction.

[0038] The frequency of rotation may be in the range 0 to 5 KHz. The upper limit of the frequency of rotation may be limited by a sampling frequency.

[0039] The two or more driving elements and the at least one sensing element are arranged in a triplet configuration comprising a sensing element provided between a first and a second driving element, optionally wherein the first and second driving elements are provided at an equal distance to the at least one sensing element. The triple configuration may comprise a first driving element, a sensing element and a second driving element,

[0040] A plurality of groupings of driving elements and sensing elements may be distributed across one or more surfaces, wherein the groupings are characterised by a spacing between driving and sensing elements and / or a relative orientation of the driving and / or sensing elements, and wherein at least one of the spacing and / or relative orientation is different between two or more groupings. The plurality of groupings of driving elements and sensing elements may be distributed across the sensing surface.

[0041] The spacing between each driving element and the at least one sensing element may be in the range between 0.1 pm to 50 pm, optionally 0.5 to 10 pm, further optionally between 1 pm and 5 pm.

[0042] The spacing between each driving element and the at least one sensing element may be at least twice the size of the object. The size of the spacing between each driving element and the at least one sensing element and the size of the driving element may be at least the size of the object.

[0043] The sensing and / or driving element may comprise at least one dimension in the range 0.3 to 15 pm. The sensing and / or driving element may be an elongate shape having a length in the range 2 pm to 20 pm and a width in the range 0.5 pm to 6 pm.

[0044] The sensing and / or driving elements may have a length in the range 2 pm to 20 pm, a width in the range 0.5 pm to 6 pm and spacing between of 1 to 5 pm.

[0045] The at least one sensing surface forms a part of a fluidic channel providing a fluidic path at or about, optionally past, the at least one sensing element.

[0046] The fluidic channel may have a size corresponding to a size of the integrated circuit

[0047] The device of any preceding claim, wherein the at least one sensing element is arranged at said sensing surface. The two or more driving elements may be arranged at said sensing surface. The at least one sensing element may be at least partially exposed at said sensing surface such that, in use, fluid is in contact with said sensing element. The at least sensing element may comprise an insulator layer such that the insulator layer is in contact with said sensing element. An insulator layer may be provided on said sensing element and the insulator layer may from part of the sensing surface and / or be exposed to the fluid. The insulator layer may comprise an insulator material, for example, SiO2,

[0048] The object may have at least one dimension, for example, a height, length or width, in the range 10 nm to 10 urn.

[0049] The object may have a dimension, for example, a height, length or width between 0.2 to 5 pm. The sensing and / or driving elements may have a length in the range 2 pm and 20 pm and / or a width between 0.5 pm and 6 pm and / or a spacing between 1 pm and 5 pm.

[0050] At least one of a length, width, spacing of the driving and / or sensing elements may be selected based on at least the size of the rotatable object. The rotatable object may be positioned, for example, by a tethering and / or attachment method. The position of the object may be limited to within a spatial variation or range by a precision of the method. At least one of a length, width, spacing of the driving and / or sensing elements may be selected based on at least the spatial variation and / or range and / or precision of the positioning method.

[0051] The object may be rotated or otherwise moved by a synthetic or natural nanometre or micron-sized rotary motor. The object may be rotated or otherwise moved by an action of a synthetic or natural nanometre or micron-sized rotary motor.

[0052] The rotary motor comprises at least one of: a bacterial flagellar motor; a F1 , Fo, Fo-ATP synthase, or equivalents for the Vacuolar-type ATPase (V-ATPase); a birefringent particle.

[0053] The at least one further property may comprise an electrochemical gradient of a cell. The at least one further property may comprise a property related to flow and / or movement of the fluid, for example, a flow rate and / or viscous load. The at least one further property may comprise a chemical property of the fluid, for example, an identity of a molecule or chemical element in the fluid.

[0054] The at least one further property may comprise a property of the fluid. The fluid may comprise a liquid.

[0055] The at least one sensing element may be arranged at said sensing surface such that the at least one sensing element is at a suitable distance to detect a response signal from said fluid contacting the sensing surface. The distance may be dependent on at least the size of the at least one sensing element, the magnitude or other property of the driving signal.

[0056] The at least one sensing elements and / or driving elements may be attached and / or embedded and / or otherwise integrated into the sensing surface.

[0057] In accordance with a further aspect, there is provided a method of sensing a rotational property and / or a further property associated with one or more objects, for example, nanometre or micron sized objects in a fluid, comprising: driving two or more driving element using two or more respective signals characterised by a difference in at least their phase and / or other electrical property; sensing a response signal in response to driving the driving element, wherein the difference in at least the phase and / or other electrical property of the two or more respective signals causes the sensed response signal to be dependent on at least a difference in impedance between each of the two or more driving elements and the at least one sensing element such that a rotational property of one or more objects in the fluid and / or at least one further property for the one or more objects or for the fluid is obtainable from the sensed response signal. The rotational property and / or at least one further property comprises a speed of rotation and / or a frequency of rotation and / or a direction or rotation and / or a change in direction of rotation.

[0058] The method may comprise positioning the object about the at least one sensing element. The method comprises positioning the object by tethering and / or attaching the object or a further attached object thereby to substantially prevent linear movement of said object and / or substantially fix a rotational axis of said object and / or to permit rotation and / or circular or elliptical movement of said object. The at least one sensing element and / or driving elements may be provided as part of or disposed in a sensing surface and the method may comprise contacting a fluid using the sensing surface.

[0059] The method may further comprise processing the sensed response signal to obtain the rotational property and / or at least one further property. The method may further comprise performing a demodulation process on the sensed response signal.

[0060] Features of any one aspect may be applied as features of any other aspect of the invention, as appropriate. For example, method features may be applied as device features and vice versa.

[0061] Brief Description

[0062] Various aspects of the invention will now be described by way of example only, and with reference to the accompanying drawings, of which:

[0063] Figure 1 is an illustration of rotation of a rotatable object due to rotation of a bacterial flagellar motor;

[0064] Figure 2(a) illustrates sensing elements for sensing an impedance between two electrodes in a solution and Figure 2(b) is a plot of the frequency dependence of a sensed impedance;

[0065] Figure 3(a) is a schematic diagram of part of a sensing device, in accordance with an embodiment, Figure 3(b) is a schematic diagram of the sensing device, and Figure 3(c) is an equivalent circuit diagram of the device;

[0066] Figure 4 is a schematic diagram of part of the sensing device of Figure 3;

[0067] Figure 5 is a perspective view of a sensing device, in accordance with an embodiment;

[0068] Figure 6(a) is a top-down view of a sensing device, in accordance with an embodiment, and Figure 6(b) is a side view of the sensing device;

[0069] Figure 7 is an image of an integrated circuit forming part of the sensing device, in accordance with embodiments;

[0070] Figure 8(a) is an image of the integrated circuit and Figure 8(b) is an image of a printed circuit board comprising the integrated circuit, in accordance with embodiments; Figure 9(a) is a plot of experimental results obtained using the device and Figure 9(b) is an image of a rotatable object, in particular a rotatable object secured to a bacterial flagellar motor;

[0071] Figure 10 depicts a first arrangement of sensing elements in accordance with an embodiment;

[0072] Figure 11 depicts alternative sensing element configurations;

[0073] Figures 12(a), 12(b) and 12(c) are images of part of the sensing device;

[0074] Figures 13(a) to (d) are further views of part of the sensing device of Figure 5;

[0075] Figure 14 is an image of the sensing device, in accordance with an embodiment; Figure 15 is a further view of the device, in accordance with an embodiment, and Figures 16(a) to (d) are plots of experimental results obtained using the device.

[0076] Detailed Description

[0077] The detection and determination of properties of an object, for example, objects embedded, suspended or otherwise contained or conveyed by a fluid is described in the following. While the following described embodiments relate to rotation of an object secured to a bacterial flagellar motor, it will be understood that other rotatable objects may be detectable using the sensing device. In the following a sensing device architecture is described that may be used for environmental and physiological sensing. The device has a bio-hybrid electronic architecture and provides a bioelectrical interface via the bacterial flagellar motor.

[0078] In the following, objects in the fluid may be referred to as rotating or rotatable object and rotational properties of said objects are also described. Rotation will be understood as movement about a point or an axis. For example, the objects may move in a circular or elliptical trajectory or orbit about an axis and the determined properties relate to said movement. The objects may be considered to revolve about an axis or point. In the following the term rotatable and rotating is used and will be understood to cover such movement. The speed of rotation and / or frequency of rotation may comprise the speed and / or frequency of moving around an axis or point. The direction of rotation may include the direction of movement about said axis.

[0079] The one or more objects may move in a closed trajectory and / or orbit. The closed trajectory and / or orbit may be about an axis and / or a point. The speed of rotation may comprise a speed of traversing the closed trajectory and / or orbit. The frequency of rotation may comprise a frequency of traversing the closed trajectory and / or orbit. The direction of rotation may comprise a direction of traversing the closed trajectory and / or orbit.

[0080] Embodiments described in the following are based on a so-called “bead assay” or a “tethered cell assay” and an integrated circuit. Figure 1(a) illustrates a bead assay and Figure 1(b) illustrates a tethered cell assay. A single motor bead assay relies on a 50 amino acid mutation to the protein FliC (the filament is composed of about 30,000 repeats of that single protein). This known mutation is called a sticky filament phenotype and enables spontaneous sticking of filaments to commercially available polystyrene particles (beads) that come in various, tightly controlled sizes, that are slightly charged to avoid sticking of beads to clumps in solution. The filament can also readily stick to glass. If the filament is shortened (by a shearing process) to a stub, the load on the motor is now imposed by a rotating bead (as show in Figure 1A).

[0081] The cell may be attached to the surface via a positively charged polymer, relying on the fact the cell body is negatively charged. The tethered cell assay turns the system upside down meaning that the FliC has sticky properties that can be used to spontaneously attach the filament, rather than the cell, to the coverslip surface. When this happens, the motor rotates the entire cell body (a shown in Figure 1 B). Because the cell body is larger (on average a spherocylinder 1 pm in diameter and 2pm in length) than most plastic beads (ranging in size between 0.2-3pm in diameter) the rotation of the cell body is slower (up to 15 Hz).

[0082] In the following, both configurations, bead assay and tethered cell assay may be used to detect rotational properties of the flagellar motor. For example, the detection may be rotation of an object that is either the bacterium cell itself or an object attached to the bacterium (for example, a plastic bead). In addition, in other embodiments, the sensing device may be capable of obtaining rotational information and properties for an object driven by a different natural or synthetic motor.

[0083] In some embodiments, the motor itself is rotating, for example, about a body axis and that rotation causes movement of an object, for example, an attached bead. That movement in general is circular or elliptical and follows a closed trajectory. The sensed response, described in the following, includes rotational information relating to movement of the attached object.

[0084] Figure 2(a) is an illustration of an impedance between two electrodes in a solution, with a bacterium between the electrodes. Figure 2(a) depicts bacterium 12, and a pair of sensing elements, in this embodiment, first sensing element 14a and second sensing element 14b. The first and second sensing elements are electrodes. It will be understood that the bacterium is provided, for example, suspended, in a solution.

[0085] In operation, the sensing elements are driven to generate an electric field next to the electrodes. The sensing element may alternatively be referred to as driving elements. In particular, an alternating voltage is applied between the electrodes, indicated by voltage 16. As a result of the application of the alternating voltage a current flows between the electrodes and the current is dependent on an impedance (Z) between the sensing elements. The presence of an object, in Figure 2(a) the bacterium itself, will provide a contribution or modify the impedance between the sensing elements

[0086] It will be understood that the impedance between electrodes is dependent on the properties of the applied electric field, in particular, the impedance is dependent on the frequency of the applied alternating voltage. Figure 2(b) is a plot 18 illustrates the signal frequency dependence of the impedance. At a first, lower, range of frequencies 19a, (between 104and 105Hertz) the impedance is dominated by a double layer capacitance effect. At a second, intermediate range of frequencies 19b (105to 107Hz) the impedance corresponds to a resistance of the solution present between the two electrodes. At a third, higher range of frequencies 19c, in this embodiment, above 107Hz, the contribution to the impedance is substantially due to the bacterium. As can be seen in Figure 2(b), the impedance due to bacteria is smaller than the impedance due to the presence of solution (Rsol). Ccell in the figures corresponds to the capacitive coupling between the electrodes, so the electrochemical cell, while the bacterium influencing solution resistance Rsol is desired.

[0087] Figure 3(a) depicts part of a sensing device or sensor, in accordance with an embodiment. Figure 3(a) depicts a sensing element configuration. In the following, the terms sensing element and driving element are used. In the embodiment of Figure 3, a sensing element and a driving elements are provided at or near a sensing surface and that the sensing element and driving elements have the same material properties and dimensions. The sensing and driving elements may be any sufficiently conducting material. For example, metals such as gold, aluminium, copper may be used. Other materials, such as graphene may be suitable. In particular, both the driving elements and sensing elements are electrodes, having a sensing part that is substantially planar. Therefore, in terms of structural and material properties, the driving element and sensing element may be substantially the same and therefore may be both referred to as sensing elements, as appropriate. In further embodiments, the driving and sensing elements may have one or more differences, for example, a structural or a material difference. However, with regard to the operation of the device, the terms sensing and driving elements are used in the following, as the driving elements are configured to be driven, for example, using a driving signal provided by driving circuitry and the sensing elements are configured to sense, for example, a sensing signal.

[0088] Figure 3(a) depicts a sensing element configuration 20, in accordance with an embodiment. The configuration of Figure 3(a) has three elements: a first driving element 24a, a second driving element 24b and a sensing element 26 arranged in a triplet configuration. In this embodiment, the sensing elements are elongate and substantially parallel. In the present embodiment, the driving elements and sensing elements are electrodes formed on a surface of an integrated circuit (IC). In this embodiment, each driving element is provided at an equal distance to the sensing element. Further details regarding the integrated circuit is provided with reference to Figures 6 to 8. The driving elements and sensing elements are provided on or at least near a sensing surface that contacts the fluid containing the rotatable objects. In the embodiment of Figure 3, the sensing elements are provided on the sensing surface and a bacterium 31 is attached at a head end to the sensing surface. The sensing surface is not shown in Figure 3(a), but will be understood to form a surface on which the sensing elements are positioned. At its tail end, a rotatable object, in this embodiment a fluorescent bead, is attached. The bead is free to rotate about an axis by movement of the bacterium. The bacterium is attached to a point on the sensing surface at its head end. As described in the following, the rotatable object rotates with a rotation frequency (fn).

[0089] In some embodiments, the sensing / driving elements have a further layer provided over their exposed surface to isolate the sensing / driving elements from the fluid. In such embodiments the further layer forms part of the sensing surface. In some embodiments, an insulating layer is provided across the sensing elements and driving elements and the insulating layer forms the sensing surface itself. As described with reference to Figure 4, the sensing surface may form part of a fluidic channel for delivering the bacterium and / or rotatable object to a sensing region by the electrodes. The insulating layer may be formed of SiO2. In some embodiments, the sensing / driving elements may be embedded in the sensing surface.

[0090] The driving elements are coupled to associated driving circuitry that include alternating current sources 28a and 28b. The driving circuitry is configured to produce a first driving signal and a second driving signal and drive the first driving element 24a and second driving element 24b with the first driving signal and the second driving signal, respectively. Driving the driving elements using a respective driving signals causes an electric field to be generated in a sensing region about the driving and sensing elements. The size of the sensing region will depend on the distance between the driving and sensing elements and also the voltage applied to the electrodes. The bead may be a distance from the surface, depending on the length of the filament. That distance may be, for example, in the range between 1 um and 50um from the surface.

[0091] The length of the bacteria may vary, however, the width may not. The bead therefore may be provided in a sensing region between 1 m and 50um away from the surface, depending on the length of the filament. In some embodiments the bacteria is tethered to a further, upper surface, and extends downwards (in a direction away from the further surface) so that the bead (or the cell) is provided closer to the sensing and driving elements.

[0092] The magnitude of the electric field may be calculated based on the voltage applied to the driving elements and may be dependent on additional factors, such as the distance between the electrodes. The generated electric field will vary over between the electrodes.

[0093] In the present embodiment, the driving signals are sinusoidal voltage signals. The first and second driving signals have an amplitude Vac. The amplitude Vac may be in the range 1 mV to 32V. The first and second driving signals have a frequency (o>) in the range 100 kHz to 100 MHz. In the present embodiment, the first driving signal is in phase relationship with the second driving signal. In particular, in the present embodiment, the first driving signal is 180 degrees out of phase with the second driving signal.

[0094] As a result of the driving of the driving elements, an electric field is formed in the sensing region by the sensing and driving elements. In some embodiments, the first driving element generates a first contribution to the electric field, referred in the following to the first electric field, and the second driving element generates a second contribution to the electric field, referred to in the following as a second electric field. The first and second electric fields cause respective first and second currents to be formed between the first driving element 24a and the sensing element 26 and the second driving element 24b and the sensing element 26. The first current flows from the first driving element 24a to the sensing element 26 and the second current flows from the second driving element 24b to the sensing element 26.

[0095] The first and second current flows are inversely proportional to first and second impedances. In particular, as depicted in Figure 3, a current between driving element 24a and sensing element 26 is equal to: where the current is inversely proportional to Zsotthe impedance of the solution between these elements. Likewise, the current between driving element 24b and sensing element 26 is equal to: where the current is inversely proportional to the sum of the impedance of the solution {Zsoi) and the contribution from the between these elements.

[0096] Due to the properties of the driving signals, in particular, the phase relationship of the first and second driving signals, the first and second electric fields will at least partially cancel.

[0097] It will be understood that, in principle, if there are / is no bacteria / um rotating between the electrodes, the differential currents from the electrodes substantially cancel each other, resulting in a zero current reading at the central sensing element when there is a 180 degree difference in phase. For other phase differences, the cancellation will not be complete.

[0098] In the embodiment of Figure 3, a bacterium is attached at a first end, at a position between the second driving element 24b and the sensing element 26. The rotatable object 30 attached to the bacterium rotates in a region between the sensing element 26 and the second driving element 24b. The rotatable object rotates at a rotating frequency fR. In the presence of the rotatable object, the second electric field is modified such that the second electric field has a contribution that is dependent on the rotating frequency of the object. Due to the positioning of the bacterium in Figure 3, the first electric field is substantially unmodified or at least modified to a lesser degree by the rotation of the rotatable object such that the first electric field does not have a contribution dependent on the rotating frequency of the object or has a lesser contribution that is dependent on the rotating frequency of the object.

[0099] The bacterium may be attached or tethered. Tethered may mean that the bacterium will be rotating too (which it might but the signal is then a combination of a rotating bacterium and the bead), while as a result of attaching the bacterium, only the bead rotates.

[0100] Driving both driving elements simultaneously causes at least a partial cancellation of the generated electric field, in particular, a cancellation of the background field from the solution while retaining the contribution produced due to the presence of the bacterium or rotatable object. For example, when the rotatable object is rotating the contribution is a rotating frequency dependent contribution.

[0101] As a result of the driving of the driving elements, a first impedance is formed between the first driving element 24a and the sensing element 26 and a second impedance is formed between the second driving element 24b and the sensing element 26. In Figure 3(a) the first impedance corresponds to the current labelled 11 and the second impedance corresponds to the current labelled I2. In the presence of the rotatable object in the sensing region, the second impedance between the second driving element 24b and the sensing element 26 has a contribution from the solution (Zso() and a contribution that is dependent on the frequency of rotation AZ( R). The first impedance between the first driving element 24a and the sensing element 26 has a corresponding, equal but opposite contribution from the solution (Zso() and substantially no contribution that is dependent on the frequency of rotation, or at least a lesser contribution from dependent on the frequency of rotation. The contribution that is dependent on the frequency of rotation (AZ( R)) may be referred to as the rotation dependent impedance. In the absence of the rotatable object, the rotation dependent impedance is substantially zero.

[0102] By sensing the response to two driving signals at a given frequency having a 180 degree shift from each other the background impedance contribution from the solution is cancelled at that frequency. Therefore, the sensed response signal is dependent on the difference in the first and the second impedance and the difference is impedance due to the rotatable object (AZ( R)) can be measured. The sensed response at the sensing element is a combination or sum of responses to each of the driving signals.

[0103] A direct measurement of AZ(K) may be attempted by detection of Zsol + Z fR). However, as Z(fR) is so small relative to Zsotthe certainty that one is detecting this contribution is low. By cancelling Zsoiusing driving signals shifted in phase by 180 degrees, the contribution Z fR) from the rotating bacterium can be detected. The driving signals thus enable a differential measurement to be performed to detect small changes in the current that are induced only by the BFM rotation. Because the impedance change due to rotation of bacteria, compared to the impedance change just due to the presence of solution (Rsol) is so small a differential impedance measurement is used. The rotating object (in this case, the bacterium with a bead) results in a fractional change in the impedance of solution. A differential measurement approach allows detection of the fractional impedance change. This is because using differential measurement allows cancellation of the large offset from the solution impedance itself.

[0104] In the above described embodiments, two driving signals having a phase difference of 180 degrees was described. The phase difference may be between 0 and 180, however, the cancellation is greatest at 180 degree, when out of phase. In some embodiments, a partial cancellation may be sufficient to allow detection of the rotation frequency dependent contribution, and therefore the phase difference may be in the region between 135 to 225 degrees, optionally between 150 to 210 degrees. The produced driving signals may be referred to as complimentary or differential signals. While Figure 3(a) depicts attachment of the bacterium between a driving element and a sensing element, it will be understood that the objects may also be attached to a surface area of the driving and / or sensing electrodes to allow detection of rotational properties. The bacterium may be positioned between the first driving element 24a and the sensing element 26. The bacterium may be positioned at an intermediate position, for example at the sensing element 26, such that both the first electric field and second electric field have a contribution from the rotatable object. Depending on the position of the cell body, a full cancellation may not be achieved. It would be achieved if phase is 180deg and everything is symmetrical. In some embodiments, the rotating object passes the area between at least two, optionally all 3 of the electrodes.

[0105] Figure 3(b) is a cross-sectional schematic diagram of the device. Figure 3(b) depicts an object, in this case a bead 30. As described elsewhere, the device may also detect properties of the cell. Figure 3(b) also depicts sensing element 26 and first and second driving elements 24a, 24b. The elements are formed on a sensing surface which forms an upper surface of an integrated chip 29. The integrated chip is formed using a CMOS manufacturing process.

[0106] Figure 3(b) shows the detection mechanism involving (i) three equally spaced electrodes (corresponding to the central sensing element 26 and the two driving elements 24a, 24b). Figure 3(b) also depicts a bacterium with bead assay located between the two outer electrodes, preferably on the middle electrode. The bead assay located preferably on the middle electrode for better signal to noise ratio and to allow a clearer detection of changes in direction of rotation.. As described with reference to Figure 3(a), two equal and out of phase signals are applied at the two outer electrodes (the driving elements) while the middle electrode senses the difference in impedance between the two pairs of electrodes as a current flowing out of it. The device is configured such that the two pairs of electrodes are identical so that substantially no net current flows out of the central sense electrode in the absence of the bacterium. Figure 15 illustrates the principle of detection of directional information.

[0107] When the bacterial cell body is positioned between the electrodes, as shown in Figure 3(a), the BFM-driven bead modulates the amplitude of the differential impedance between the pairs of electrodes periodically with a rotation frequency fr. In some embodiments, this amplitude modulation results in a current flowing out of the middle electrode that is a function of both the rotation frequency frand the frequency f of the sinusoidal excitation signals used to drive the pairs of electrodes. A demodulation process is then performed to obtain the rotational information, in this embodiment, the rotational frequency. The resulting current, when converted to a voltage and then demodulated with a signal at the frequency f by an integrated circuit, yields a sinusoidal output signal at the rotation frequency fr. The current flowing out of the middle electrode is the input signal amplitude-modulated by this periodic signal of rotation frequency frThis current when demodulated by the integrated circuit will give a sinusoidal function having frequency fr.

[0108] It will be understood that the rotating bead follows a trajectory or orbit. The phase of the signal detected at the sensing element is dependent on the position of the bead on this trajectory. As such, a change in direction of rotation is detectable as a sudden phase change in the detected sinusoidal signal. Further description on detection of a change in direction is provided with reference to Figure 15.

[0109] In some embodiments, the device is also configured to detect a rotation of the cell body itself, where the filament is attached in between two electrodes, and ideally in the middle of the third electrode, so that the body rotates.

[0110] Continuous monitoring of the localized impedance changes within a microscale sensing site allows for electrically detecting the rotation speed and direction of the bacterial flagellar motor.

[0111] As depicted in Figure 2(b), the impedance between each of the two pairs of electrodes comprises a double-layer capacitance (CDL at the electrode solution interface), the solution resistance and capacitance (RSOL and CSOL), and the parasitic coupling capacitance between the electrodes (CCELL; note ‘cell’ refers to electrochemical cell here). At low frequencies, up to about 100kHz, the equivalent impedance between the electrodes is dominated by CDL, making the detection of small changes in the solution impedance produced by the bead rotation impractical. At high frequencies above about 10MHz, the equivalent impedance between the electrodes is set by the impedance of CCELL, which also prevents the detection of the impedance changes produced by the bead rotation. To be able to properly measure the impedance changes due to the bead rotation, the operating frequency must be in the frequency range from about 100kHz to about 10MHz, where solution impedance dominates.

[0112] Figure 3(c) depicts a schematic circuit diagram showing different parts of the device as electrical components, as an equivalent circuit. The two terminals on the left correspond to the driving electrodes where the two driving signals Vac(com) and - Vac(com) of 180° phase difference is applied. The terminal on the right corresponds to the sensing electrode into which the differential current flows (the current Al). The first impedance between the first driving electrode and the sensing electrode and the second impedance between the second driving electrode and the sensing electrode is the same and includes contributions from double layer capacitances (CDL) at the electrode solution interfaces, the solution resistance (RSOL) and solution capacitance (CSOL). When the bacterium is positioned on the sensing electrode and the bead attached to its flagella rotates about it, the impedance between the both sets of electrodes is periodically altered by the rotating bead with a frequency (labelled by rotational frequency cor). The differential current flowing from and sensed by the sensing electrode therefore is dependent on the input signal of frequency com and the rotation frequency cor. The detected signal can be processed to obtain the frequency of rotation and other rotational properties, for example, the direction of rotation or a change in direction of rotation.

[0113] As part of the sensing process, a frequency for the driving signal is selected as 10 MHz. A response signal, in this embodiment, a current, is sensed by the sensing element 26 and processed by response signal detection circuitry, as described in further detail in Figure 4. In operation, a response signal, in this embodiment, a current, is measured at the sensing element 26. The measured current is dependent on the frequency of the driving signal co. The measured current is also dependent on the contribution to the impedance caused by the rotating object, as described above. As such the measured current is dependent on the frequency of rotation. The measured current is also dependent on the voltage of the driving signals. As described in further detail with reference to Figure 4, the response signal is processed by response signal processing circuitry to obtain the rotation dependent impedance. From the rotation dependent impedance, rotational information, in particular, one or more rotational properties may be obtained. Figure 4 depicts detection circuitry for the sensing device, in accordance with an embodiment. Figure 4 depicts the sensing element configuration 20 as described with reference to Figure 2 together with response signal detection circuitry. The response signal circuitry has a first module 42 for converting current to voltage. The first module is configured to receive the sensed current signal, as described with reference to Figure 2, and convert the sensed current signal to a voltage signal. The second module 44 is configured to receive the voltage signal from the first module and perform a demodulation process on the voltage signal to produce two voltage components (Vreaiand Vimag).

[0114] The first module 42 has a current amplifier 46 with a programmable gain and a transimpedance amplifier (TIA) circuit configuration 50 having a transimpedance amplifier 52. The sensed current is provided to the current amplifier 46 to produce an amplified current. The amplified current output from the current amplifier 46 is provided to the TIA circuit configuration to be converted to a voltage by the transimpedance amplifier 52. The gain of the TIA amplifier is set by the value of Rf. The values of these components enables control of the gain of the electronics chain and hence the output voltage amplitude.

[0115] The second module 44 is a demodulation module and has an oscillator 54, a doublebalanced passive mixer (52a, 52b), a unity gain buffer (not shown) and Gm-C low-pass filters (56a, 56b) for demodulation. The second module 44 is configured to obtain and output in-phase and quadrature electrical signal components from the voltage output by the current to voltage module. The second module 44 allows the extraction of the real and imaginary parts of the voltage thus allowing determination of the impedance.

[0116] While Figure 4 depicts an example of detection circuit, in accordance with embodiment, it will be understood that, in other embodiments, other suitable circuitry can be provided to convert the response signal into an impedance signal.

[0117] In the present embodiment, the output of the detection circuitry is provided to a processing resource (not shown), via a sensing device signal readout module. In the present embodiment, the processing resource is processing circuitry configured to process the sensed electrical response to determine a rotational property of the rotatable object or an attached rotary motor or a further property of the fluid based on the rotational property of the rotatable object or attached rotary motor. For example, the rotation property may be a rotational speed of the rotating object or a rotational frequency of the rotating object. The obtained frequency of rotation may be in the range 0 to 5 KHz. However, it will be understood that the upper limit of the detectable frequency of rotation may be set by the detector apparatus.

[0118] In the above described embodiment, the frequency for the driving signals is selected as 10 MHz, however, it will be understood that alternative suitable frequencies may be used. For example, the signal frequency may be in the range 100 kHz to 100 MHz. In some embodiments, a range of frequencies may be swept over such that the sensed signal is dependence on the range of frequencies.

[0119] The processing resource may be further configured to determine a further property of the fluid based on the rotational property. For example, a property related to flow and / or movement of the fluid, for example, a flow rate and / or viscous load may be determined. Alternatively, a chemical property of the fluid, for example, an identity of a molecule or chemical element in the fluid may be determined. In some embodiments, the sensing device forms part of a mechanosensor or a chemosensor.

[0120] Figures 3 and 4 depict the electronic sensing module of the sensing device. The solution that contains the rotatable objects must be provided in a suitable sensing region at or adjacent to the sensing elements. As described above, the positioning of the bacterium relative to the sensing elements may have an effect on the obtained signal. Random positioning of the bacteria suspended in liquid may decrease the chance of the bacteria settling close to or onto the gap between an electrode pair of interest among the three sensing electrode which may lead to a degradation in the repeatability of the measurement. To improve the repeatability, a different configuration based on a channel and specific attachment may be used that allow positioning of the bacteria with the rotating motor in a sensing region. The sensing region may be adjacent to or next to the electrode pair of interest. In some embodiments, as described above, the bacterium may be attached to a surface other than the sensing surface so that the rotatable object is in a sensing region next to the sensing surface. A fluid delivery apparatus formed using PDMS is described in the following to control the liquid flow and enable perfusion. The apparatus has a channel that runs over and passes over the electrode area and is connected to the inlets using two layers formed using PDMS patterning. As described in the following, the PDMS structure protects all other electronics in the chip and provides space for the wire bonded area. In the following embodiments, a PDMS structure is described, however, alternative materials and techniques may be suitable, for example, glass etching.

[0121] Figure 5 depicts a view of a fluid delivery module or mechanism forming part of the sensing device for delivering the rotatable object to the sensing region. The fluid delivery module has a layered structure comprising: a printed circuit board 102 and one or more microfluidic layers. In the embodiment of Figure 5, there are two microfluidic layers: first microfluidic layer 104a and second microfluidic layer 104b. Disposed between the printed circuit board and the microfluidic layer(s) is an integrated circuit 108 having the sensing and driving element configuration. The microfluidic layers will be understood to be formed from Polydimethylsiloxane (PDMS), in the present embodiment. The microfluidic layers define a fluid delivery arrangement for delivering fluid to the integrated circuit 108, in particular to the sensing elements of the integrated circuit 108.

[0122] The device also has an inlet 110, in this embodiment an inlet tube 110 and an outlet 112, in this embodiment an outlet tube 112. The inlet is for receiving a fluid and the outlet is for removal of the fluid. The microfluidic layers have channels formed therein to define a fluidic path between the inlet and the outlet. The fluidic path formed between the inlet and the outlet will have a sensing portion. In the present embodiment the fluid delivery module includes a fluidic channel defined through the microfluidic layers and defining a fluidic path from the inlet to the outlet. In the present embodiment, the fluid delivery arrangement is defined to deliver fluid to the integrated circuit.

[0123] The inlets and outlets may be referred to as reservoirs. These will, generally, have dimensions much larger (for example, mms to cms) compared to the size of the bacteria. The microfluidic device therefore has a first layer that receives the fluid and a second layer that delivers fluid to the integrated circuit. The two layer structure bridges the cm scale of the reservoir and the urn or mm scale of the integrated chip.

[0124] The gap between the mm scale of the integrated circuit (in current embodiment 600um, it can be increased up to 2mm) and the cm scale of the general fluid handling systems, for example, the reservoirs is bridged by the two layer structure. It will be understood that the size of the bacteria is not of concern to the microfluidics. The microfluidics has to fit into this mm scale area so it does not damage the wire bonds at the periphery of the IC. It has to provide the bacterial solution to the urn scale sensor area on the IC while sealing the electronics around it from fluids. As such, as least one dimension of the fluidic channel is selected to match the size of the sensing surface or integrated chip.

[0125] In further detail, the fluidic channel has a first input portion 114, a second input portion 116, a sensing portion 118, a first output portion 120 and a second output portion 122. The channel is such that fluid passes through the first input portion 114, the second input portion 116, the sensing portion 118, the first output portion 120 and the second output portion 122 in order. In the present embodiment, the first input portion 114 and the second output portion 122 are formed in the second microfluidic layer 104b and are substantially horizontal. In the present embodiment, the first input portion 114 and second output portion 122 are parallel and, in particular, co-linear and are provided at a first height corresponding to an external, upper surface of the device. In the present embodiment, the sensing portion 118 is formed in the first microfluidic layer 104a and is parallel to the first input portion 114 and second output portion 122 but provided at a different, lower height. In the present embodiment, the second input portion 116 and the first output portion 120 are formed in the first microfluidic layer 104a are substantially vertical and parallel thus joining the channel portions at the first height to the sending portion at the lower height.

[0126] The sensing portion 118 is a fluidic channel that passes the sensing elements of the integrated chip 108. The sensing portion therefore has as its lower surface, a sensing surface for contacting the fluid. A surface of the sensing portion, for example, the sensing surface or a further surface may be patterned to control and to reduce and / or prevent linear movement of the bacterium as described with reference to Figure 12. The attaching and / or tethering may further fix a rotational axis of the rotatable object and / or permit rotation of said rotatable object.

[0127] The sensing portion is a fluidic channel and has a lower surface, an upper surface and two side surfaces. In some embodiments, the patterning may be provided on one or more of these surfaces. In some embodiments, the cell may be attached or tethered to the upper surface so that the rotatable object of interest adjacent to the sensing surface. While Figure 5 depicts a fluid delivery module in accordance with an embodiment, in other embodiments, alternative fluid delivery arrangements and configuration may be provided. For example, one or more of the input or output portions may be formed in the same microfluidic layer as the sensing portion. Likewise, the vertical portions may be not vertical, in some embodiments. Likewise, the horizontal portion may not be horizontal, in some embodiment. In a non-limiting example, a diagonally sloping channel may be provided.

[0128] Figure 6(a) depicts a top-down view of the sensing device. Figure 6(a) depicts the microfluidic layer(s) 104, the inlet 110 and the outlet 112. The fluid delivery arrangement is also depicted and, for clarity, only the sensing portion 118 is labelled. As depicted in Figure 6(a), the integrated chip 108 has an electrode area and a wire bonded area.

[0129] Figure 6(b) depicts a side view of the sensing device. The side view is a cross-sectional view along X-X’ of Figure 6(a). Figure 6(b) depicts PCB layer 102, a first microfluidic layer 104a and second microfluidic layer 104b and the integrated chip 108. Figure 6(b) depicts the sensing portion 118 of the fluid channel. Figure 6(b) also depicts the position of the electrode area 124 of the integrated chip and the wire bonded area 126. As can be seen in Figure 6(b), the wire bonded area 126 corresponds to a portion or cavity formed under the second microfluidic layer 104b and beside the first microfluidic layer 104a. The wire bonded area 126 is adjacent to the integrated circuit 108 and the printed circuit board 102. The first and second microfluidic layers form a barrier between the wire bonded area 126 and the fluidic channel 118, such that, in use, the wire bonded area is isolated from the fluid. Figure 5 depicts a top view and a side view of a sensing device. The side view shows the channel running over the electrode area, and structures to accommodate the wire bonded area and inlets to introduce the bacteria solution. As depicted in Figure 6(a) the integrated chip has first and second wire bonded areas and an electrode area.

[0130] While Figures 5 and 6 depicts a fluid delivery module forming part of the sensor, in accordance with embodiments, alternative fluid delivery modules and mechanism may be suitable. In some embodiments, a solution may be dropped onto a sample surface. For example, in some embodiments, the electrodes are exposed to liquid (1-3 J) that contain bacteria and a coverslip is placed on top of the liquid to prevent liquid evaporation. Figure 7 is a schematic design of the IC with the integrated sense electrodes, fabricated in a TSMC 0.18 UM CMOS High Voltage Mixed Signal based LDMOS FSG Al 1 P6M 1 .8 / 5 / 32V process. The red box indicates the area where the sensing microelectrodes are located. There are 64 electrodes with different sizes and gaps between electrodes (all on the order between 1 to 10 pm) in this design. As depicted in Figure 7, the integrated chip has an electrode area 204 and an electronics area 202. The integrated circuit board is encapsulated with a UV curable epoxy (except the region where the sense electrodes are located), to insulate the chip from the environment as the epoxy is resistant to moisture. While Figure 7 depicts 64 electrodes, it will be understood that a larger number can be incorporated. As a non-limiting example, at least 1000 electrodes may be provided. Figure 11 depicts a number of electrode configurations.

[0131] Figure 8(a) is an image 302 of the integrated circuit wire bonded to the printed circuit board, in accordance with an embodiment. The wire bond and the electronics must not be exposed to liquid. Therefore, a shielding layer of epoxy is deposited to shield the integrated circuit chip except the electrodes and the extent of the printed circuit board covered by the wire bonds. Figure 8(b) is an image 304 of the printed circuit board, in accordance with an embodiment. The integrated circuit 306 is marked. Figure 8(b) further shows the components forming the driving circuity for applying the ac signals to electrically drive the electrodes of the IC and the detection and demodulation circuit and demodulation. Further readout circuitry is also provided to allow readout of output data from the chip. Configuration circuitry is also provided on the printed circuit board to configure the chip setting. In this configuration, the bacteria with rotating motors randomly settle next to one of the electrodes, and we are able to detect the rotation of the bead or the cell body. Figure 8(b) shows the PCB with the IC 306 indicated by a black circle.

[0132] Figure 9(a) is a plot of experimental results obtained using the device and Figure 9(b) is an image of a rotating object. Figure 16(a) to (d) below depict further experimental results. These results are obtained using different bacteria and different size markers. The results of Figure 9 and Figures 16(a) to (d) depict results obtained using detection of an electrical signal and compared to known optical detection techniques. These known optical techniques expose a sample to a blue light is at a power that causes damage to the bacterial membrane and slows down the motor. Depending on when the sample is exposed to such light the optical signal is expected to be a bit slower or faster than the electrical. In contrast, the electrical signal is detected using the device without exposing the sample to a blue light.

[0133] Figure 9(a) depicts a plot having frequency (Hz) as the x-axis 902 and amplitude of electrical signal as the y-axis 904. Figure 9(a) has a vertical line indicating the detecting rotation frequency (detected using an optical method) at 0.24 Hz.

[0134] Figure 9(a) depicts a first measured electrical signal without bacteria 908 and a second electrical signal with bacteria 910. Each signal is dependent on frequency and may be referred to as a frequency spectrum. A rotation frequency peak 912 is detected from the second electrical signal at 0.32 Hz. The peak in the frequency spectrum of electrically detected signal close to speed of visual rotation of BFM rotation marker on that electrode.

[0135] The signal 908 is a control measurement of an average frequency spectrum from several measurement using the device without a bacteria present. In grey, is the standard deviation of the noise in these electrical measurements without bacteria. The ratio of the amplitude of the signal with bacterium with respect to without bacterium peaks at 0.32Hz. This frequency is close to the optically observed frequency of 0.24Hz on the left. It has been proposed that the BFM speed reduces with exposure to the light used for viewing the marker, causing a speed reduction. The signal to noise ratio of this detected electrical signal at 0.32 Hz is 11 ,5dB.

[0136] Figure 9(b) is a frame of a video sequence of a rotating marker captured by an image capture device, specifically a CCD camera, images are taken with a standard microscope Figure 9(a) depicts the rotating marker 914 and the sensing element 1626 together with two driving elements 924a, 924b on opposing sides in a triplet configuration. The image is an optical recording of a bacterium with rotating BFM marker positioned on the centre electrode of a 3-electrode set. It is rotating approximately at a frequency of 0.24Hz.

[0137] 7

[0138] In other embodiments alternative quantities may be obtained. For example, a power spectrum that was obtained from the measured output voltage from the electronics using signal post-processing. The signal is the power spectrum of the real / imaginary part In the above embodiments, a single set or grouping of electrodes, in particular a triplet of three electrodes was described. In some embodiments, a plurality of electrode grouping are distributed across a sensing surface on a single chip. Figure 10 depicts an example array 502 of groupings of electrodes distributed across a sensing surface. An example triplet configuration of electrodes 504 is labelled in Figure 10.

[0139] By providing an array of groupings, an averaging of signals from multiple sensing may be performed in real time. Additional rotational properties may also be detected by using a plurality of groupings, for example, an ability to detect heterogeneities in rotation caused by micro-environmental differences (for example, plumes and gradients) may be provided by a plurality of groupings. In addition, the groupings may allow large data samples from large numbers of cells for analysis to be collected. The groupings of electrodes may be integrated on a single chip. There may be a large number, as a nonlimiting example, in the region of 100s, potentially 1000s, of sensing electrode arrays on a single chip. The measurement using an array will enable averaging of the signals from multiple electrodes in real-time for more robust sensing, Figure 10 is a schematic of an array of electrode sensors for detecting the rotation speed of several bacteria in parallel. In some embodiments, the detected rotation speeds or other properties are displayed on a display of a computing apparatus.

[0140] While Figure 10 depicts an array in which a spacing between electrodes for each grouping and a spacing between respective groupings are both uniform, in further embodiments either of these spacing parameters may be varied. In addition, the orientations of the groupings are also uniform such that the electrodes are arranged in a parallel arrangement. The relative orientations of each grouping and / or the orientation of one or more electrodes in the grouping may be varied between groupings. Likewise, the size and / or shape of the electrodes, for example, the surface area of the electrodes may also be varied.

[0141] Figure 11 depicts examples of groupings of electrodes, in accordance with embodiments. The device may include groupings having different properties (for example, a spacing or relative orientation). Figure 11(a) is labelled to depict sensing element 626 and first driving element 624a and second driving element 626b of a triplet. In some embodiments, the electrodes are distributed across the sensing surface, and each grouping is characterised by a spacing between driving and sensing elements and / or a relative orientation of the driving and / or sensing elements, and wherein at least one of the spacing and / or relative orientation is different between two or more groupings. Figure 11 depicts examples electrode configurations, in particular, sizes of electrodes in a triplet and the spacing therebetween. It will be understood that the size of the electrode corresponds to the surface area and is provided in terms of a width and a length. In Figure 11 each triplet is characterised by the width and length of the electrode and the spacing between the electrodes, as follows:

[0142] (a) each electrode is 10 micron by 10 micron and the spacing between each electrode is 2 micron;

[0143] (b) each electrode is 2 micron by 5 micron and the spacing is 8 micron;

[0144] (c) each electrode is 2 micron by 5 micron and the spacing is 4 micron;

[0145] (d) each electrode is 2 micron by 5 micron and the spacing is 2 micron;

[0146] (e) each electrode is 0.75 micron by 10 micron and the spacing is 4 micron;

[0147] (c) each electrode is 2 micron by 2 micron and the spacing is 2 micron;

[0148] (d) each electrode is 1 micron by 1 micron and the spacing is 2 micron;

[0149] (e) each electrode is 0.75 micron by 0.75 micron and the spacing is 1 micron.

[0150] In accordance with embodiments, the spacing between the electrodes is in the range 0.1 micron to 50 micron, optionally 0.5 micron to 10 micron. In accordance with embodiments, the electrodes have a length and / or a width in the range 0.3 to 15 micron.

[0151] As described above, a tethering means for tethering the rotatable object to, for example, is provided. In some embodiments, attachment means are provided for attaching the rotatable object. In some embodiments, polymer patterns are used for attachment or tethering to enable precise positioning of bacterium. For example, the patterns may allow the positioning of the rotatable object between a driving element and a sensing element In such embodiments, positively charged polymers, for example, poly-L-lysine (PLL), polyethyleneimine (PEI) or CellTak (RTM) are stamped onto the chip surface using poly- di-methyl-siloxane (PDMS) stamps. This would allow precise positioning of the bacteria above specific electrodes on the chip and multiplexing. Figure 12 depicts example PLL patterns achieved using PDMS stamping. It will be understood that the PLL patterns are provided along a suitable sensing surface, for example, as part of a channel, for example, the sensing channel portion depicted in Figure 5. In some embodiments, micro-printing techniques are used to create bonding sites. Figure 12(a) shows a first view 702 of a first pattern comprising 50 micron squared squares of adhesive material stamped onto a surface. The squares are arranged in a rectangular array and are spaced 100 micron apart. The array includes a first rectangular array having a size of 3 x 8 and a second rectangular array have a size of 3 x 8 squares. View 704 depicts a first close-up view of 9 squares and view 704 depicts a closer view of two squares.

[0152] Likewise, Figure 12(b), shows a first view of a second pattern comprising 3x5 pm squares. The squares are spaced 10 micron apart. The pattern is printed on the sensing surface. View 804 is a first close-up view and view 806 is a second close-up view.

[0153] Figure 12(c) depicts an image 902 of E-coli attached to the pattern. The array includes a first rectangular array having of 3 x 8 squares and a second rectangular array of 3 x 8 squares. Images 904 and 906 depicts fluorescent light from the E-coli. The pattern enables precise positioning of the bacterium at the electrodes. In further embodiments, patterning may be achieved using alternative techniques. For example, rather than via positive polymers, specific covalent attachment and genetic engineering of the bacterial strains may be used.

[0154] Figure 13 depicts technical drawings of the fluid delivery apparatus, in accordance with an embodiment. Figure 13(a) is a perspective view of the apparatus. Figure 13(b) is end elevation view of the apparatus. Figure 13(c) is a side elevation view of the apparatus and Figure 13(d) is a top-down view of the apparatus.

[0155] Figure 14 is a view of the device. Figure 14 depicts the PCB 1102, the integrated chip 1108, first PDMS layer 1103 and second PDMS layer 1104 substantially as described with reference to Figures 5 and 6. In addition, Figure 14 depicts a PDMS support layer 1101 provided between the PCB 1102 and the first PDMS layer 1103. Figure 14 also depicts needle and tubing 1128 for providing fluid to the inlet and for removing fluid from the outlet.

[0156] In the above embodiments, tethering and / or attaching means in the form of configurations of tethering and attaching sites are described. These sites are aligned with the distributions of electrodes on the sensing surface to ensure that a signal containing rotational information is obtained. For example, Figures 9, 10 and 12 depict configuration of tethering and / or attaching sites in the device.

[0157] In the above described embodiments, the rotatable object is a bacterium or an object secured to the bacterium, both being rotated by the BFM. In general, the sensing device may be used to detect rotation of other types of rotating objects. For example, an object rotated or driven by a synthetic or natural nanometre or micron-sized rotary motor may be capable of being detected.

[0158] As non-limiting examples, an object driven by a rotary motor such as F1 , Fo, Fo-ATP synthase or equivalents for the Vacuolar-type ATPase (V-ATPase) and / or a birefringent particle may be detected using the sensor. In general, rotatable objects described above may have a size in the range 1 nm to 100 microns, optionally, between 10 nm and 10 microns.

[0159] In the above-described embodiments, two driving signals having substantially the same frequency and amplitude were described. It will be understood that a different frequency and / or amplitude may be used to drive the driving elements, however, the ideal conditions for a differential measurement is to use the same frequency and amplitude.

[0160] In the above embodiments, a sensing element configuration of a triplet of electrodes is described. It will be understood that in other embodiments, more than 3 electrodes may be provided to allow detection of rotation. The limit on the number of electrodes may be based on the size of the rotating object (for example, objects may be between 1 nm and 100 microns, as described above). The limit on the number of electrodes in a group may be dependent on a combination of the size of the electrode and the object. In such embodiments, the group of electrodes are configured to be driven with electrical properties such that the produced electrical fields partially cancel at a sensing element thus allowing detection of a response signal that has a rotational frequency. In some embodiments the object is rotating asymmetrically about the sensing electrode with respect to all the driving electrodes.

[0161] Figure 15 depicts, in further detail, the operating principle of the driving and sensing elements. Figure 15(a) depicts, as described above, two driving elements provided together with a single sensing element. A rotatable object is also shown. It will be understood that the rotatable object rotates about an axis of rotation and it depicted in four positions about that axis.

[0162] As described above, two driving signals are provided to the first and second driving elements, respectively. In the embodiment of Figure 15, these are 180 degrees out of phase and therefore represented by Vac and -Vac. A signal proportional to a change in current is measured at the sensing element (Al). This signal is dependent on the movement of the object, for example, the speed and / or frequency of the object. In more detail, the sensed signal is a modulated signal dependent on the driving signal frequency and the frequency of rotation. Al is the signal obtained after the demodulation process.

[0163] Figure 15(b) depicts, a resulting graph of detected Al. As shown in Figure 15(b), the corresponding detected signal at the four positions are shown. It is observed that the detected signal is sinusoidal. The detected signal is the difference between 11 and I2 and can be represented as proportional to a sinusoidal function having an angular frequency (or angular speed) of the rotating object. The frequency of rotation or rotational frequency (f) may be determined from this angular frequency using the relation = 2n f.

[0164] In this embodiment, the bacterium is attached such that the rotation axis is normal to the sensing element and centred on the sensing element (i.e. equidistant from both driving elements). The resulting detected signal is therefore a sinusoidal signal. The rotation frequency can be detected from the signal. In addition, the direction of rotation can be detected from the signal. For example, an initial direction of current change (positive or negative) will determine if the object is rotating clockwise or anticlockwise.

[0165] To detect direction of rotation, positioning of the rotation axis in a substantially central region is required. In particular, the object should be attached and / or tethered such that the object moves in a region that is defined between the two driving elements, in particular, on either side of the central sensing element. Viewed from above, the moving object can be considered to define an orbit or closed trajectory about a central point corresponding to the axis of rotation. The orbit may be circular, as shown in Figure 15(a) or may be elliptical or an irregular shape. An orbit will be understood as at least partially curved path traced out by rotation of the rotating object. In Figure 15(a) the orbit is a circle. To detect a direction of rotation, it is preferred that at least part of the orbit of the rotation object is between the first driving element and the sensing element and at least part to the orbit of the rotating object is between the second driving element and the sensing element. The signal obtained when an orbit of the rotating object is between a single driving element and the sensing element may not provide sufficient information to obtain the direction of rotation.

[0166] A change in direction can be obtained using a number of different signal processing methods. As a non-limiting example, a detection of a discontinuity in the derivative of the signal may correspond to a change in direction of rotation. In some embodiments, the sensing device is therefore configured to obtain a derivative of the signal and identify discontinuities in the derivative.

[0167] An absolute direction of rotation of either clockwise or counter-clockwise may be obtained, for example, using detected changes in rotation. It is known that cells tend to rotate counter clockwise in the absence of analytes. The direction of rotation is therefore obtained in response to identifying a change,

[0168] Periods spent rotation clockwise are much shorter than periods spent rotating counter clockwise. Therefore, the direction may be obtained by identifying periods between changes and identifying shorter periods (for example, below a threshold) as counter clockwise and longer periods (for example, above a threshold) as counter clockwise. Alternatively, if rotation continues without a change in direction for a minimum amount of time, the rotation direction may be labelled as counter clockwise, and updated in response to changes in rotation direction. Alternatively, the initial rotation direction may be assumed to be counter clockwise and then updated in response to changes in direction.

[0169] In the embodiment of Figure 15, the orbit is equally divided between a first sensing region (formed between the first driving element and the sensing element) and a second sensing region (formed between the second driving element and the sensing element). However, such a division may not be equal, as long as at least part of the orbit passes through each of the two sensing regions.

[0170] The sensing device, as described above, may provide a response time and sensitivity up to seconds and down to nM concentrations. The relevant output for a given sensing modality is either the motor's rotational speed or direction. The latter is the output when, for example, sensing analytes, and the former is the output when, for example, sensing bacterial physiology.

[0171] The device described above, may allow measurement of the rotation of the BFM electrically, in individual cells and with high throughput. As described above, the rotation of a flagellum driven bead perturbs the electric field, thus changing the impedance measured by the electrodes. To achieve sufficient signal to noise the invention uses differential electrochemical impedance measurements, rather than simply having to handle differential impedance. For this purpose, at least three electrodes are needed to get information on the rotational direction of the motor. As described above, the rotating bead or a cell then needs to be placed over the third electrode, ideally in the middle of it. The frequency of rotational direction changes, changes in response to the presence of analytes. The design may also increase the frequencies across which spectroscopy is performed (into the MHz range), significantly increasing significantly increasing the signal-to-noise ratios and the sensitivity with which the motor rotation is detected.

[0172] Figures 16(a) to (d) are plots of further preliminary experimental results obtained using the device.

[0173] Figure 16(a) depicts a plot showing the detected electrical signal (frequency spectrum) 1708a obtained without bacteria and a second electrical signal (frequency spectrum) 1710a obtained with bacteria. An optically observed frequency 1706a of 0.4 Hz is indicated on Figure 16(a). The measured second electrical signal 1710a has a peak 1712a at 0.2 Hz.

[0174] Figure 16(b) depicts a further plot showing the detected electrical signal (frequency spectrum) 1708b obtained without bacteria and a second electrical signal (frequency spectrum) 1710b obtained with bacteria. An optically observed frequency 1706b of 0.4 Hz is indicated on Figure 16(b). The measured second electrical signal 1710b has a peak 1712b at 0.2 Hz.

[0175] Figure 16(c) is at plot showing a detected electrical signal (frequency spectrum) 1708c obtained without bacteria and a second electrical signal (frequency spectrum) 1710c obtained with bacteria. A measured optical signal 1715c is also depicted in Figure 16(c). The measured optical signal has a peak 1706c at 1.7 Hz. The measured second electrical signal 1710c has a peak 1712c at 1.1 Hz.

[0176] Figure 16(d) is at plot showing an electrical signal (frequency spectrum) 1710d obtained with bacteria. A measured optical signal 1715d is also depicted in Figure 16(d). The measured optical signal has a peak 1706d at 2.3 Hz and the measured electrical signal 1712d has a peak 1713d at 1.2 Hz.

[0177] Figures 16(c) and (d) include an optical trace in which the rotating marker was visible and a frequency spectrum of the optical trace is plotted.

[0178] Figures 16(a) to (d) show the frequency spectra of the electrical signals from electrode sets on which a rotating marker was optically observed in red, and from the same electrode set without bacteria in black (with grey showing the standard deviation of the noise - indicated by reference numerals 1718a, 1718b, 1718c). Comparing the electrical signal amplitudes on the y-axis at each of the frequencies on the x-axis, peaks in the ration of amplitude of the measured electrical signal (with bacteria) to the standard deviation were identified.

[0179] In all instances, the electrically detected signal peak frequency is close to the optically observed frequency of rotation, being slightly more or less depending on whether the electric measurement was made before or after optically observing the rotation, and how long before or after, respectively.

[0180] Known methods of identifying a peak of the spectrum may be used to determine the desired frequency of rotation from the electrical signal. For example, a fastest Fourier Transform algorithm may be used to detect the peak. The detected peaks correspond, in this case, to the rotation frequency and hence rotation speed of the BFM.

[0181] Without being bound by theory, it is known that the BFM is powered by the electrochemical potential of protons, built across the membrane it sits in. The rotational speed of the motor is proportional to that gradient. The electrical potential is due to the charge concentration difference in a close vicinity of the membrane, and the chemical concentration gradient is the entropic term. For that electrochemical potential to be maintained the membrane needs to act as a capacitor, which it does. When it is exposed to light the membrane is peroxidised resulting in holes in the membrane reducing its capacitor capability. The result is the electrochemical potential of protons is gradually reduced and so is the speed.

[0182] The following non-limiting comments are provided regarding considerations on determining design parameters of an integrated circuit of the device. In particular, factors such as size of rotatable object, precision of placement of the rotatable object are taken into consideration when considering the design parameters. As a non-limiting example, markers having a size between 1 and 5 pm were used. Without limitation, it was found that, in certain applications, larger markers may decrease the speed of rotation in higher electrical noise regimes. Electrodes having a length (L) less than 10 pm were designed to enable a larger percentage area of the electric field from them to be blocked by the 1- 5 pm markers. However, at present, bacteria may be patterned and positioned with a spatial precision in the range of 10-25 pm. Based on the precision of patterning / positioning an integrated circuit having electrodes with length about 10 pm was designed. For a marker size of 1 to 5 pm, it was found that an electrode arrangement with elongated electrodes having a length of between 2 pm and 20 pm, with width between 0.5 pm and 6 pm and spacing between 1 pm and 5 pm between adjacent electrodes offers optimal detection.

[0183] It was found that SNR obtained with an integrated circuit, in accordance with an embodiment, is 8.7 to 11.5dB. In accordance with an embodiment, an integrated chip having at least +20dB improvement through an electrical design, and additional improvements from optimising the detector / marker geometries as described above, may bring the SNR to > 40dB.

[0184] The above description of specific embodiments is made by way of example only. A skilled person will appreciate that variations of the described embodiments may be made without departing from the scope of the invention.

Claims

CLAIMS1. A sensing device for sensing a rotational property and / or a further property associated with one or more objects, for example, nanometre or micron sized objects in a fluid, the device comprising: two or more driving elements and associated driving circuitry configured to drive said driving elements using two or more respective signals characterised by a difference in at least their phase and / or other electrical property; at least one sensing element configured to sense a response signal in response to driving the driving elements using the two or more signals, wherein a difference in at least the phase and / or other electrical property of the two or more respective signals causes the sensed response signal to be dependent on at least a difference in impedance between each of the two or more driving elements and the at least one sensing element such that a rotational property and / or at least one further property associated with the one or more objects is obtainable from the sensed response signal, wherein the rotational property and / or at least one further property comprises a speed of rotation and / or a frequency of rotation and / or a direction of rotation and / or a change in direction of rotation.

2. The sensing device as claimed in any preceding claim, wherein the object is attached to a bacteria flagellum and the object rotates and / or exhibits rotational and / or substantially circular or elliptical motion due to the rotary motor of the bacteria flagellum.

3. The sensing device as claimed in any preceding claim wherein the sensing device comprises an attachment and / or tethering means on a sensing surface or on a further surface for attaching or tethering at least part of the one or more objects or a further object attached to the object.

4. The sensing device as claimed in any preceding claim, wherein the attachment and / or tethering means substantially position the object at a position at or around the at least one sensing element.

5. The sensing device as claimed in any preceding claim, wherein the attaching and / or tethering means are configured to attached and / or tether the object thereby to substantially prevent linear movement of said object and / or substantially fix a rotationalaxis of said object and / or to permit rotation and / or circular or elliptical movement of said object.

6. The sensing device as claimed in claims 3 to 5, wherein a first sensing region is defined between the at least one sensing element and the first driving element and a second sensing region is defined between the at least one sensing element and the second driving element and wherein the attachment or tethering means attach and / or tether the object such that the object follows a path in at least part of both the first and second sensing regions.

7. The attachment and / or tethering means may comprise one or more surfaces comprising one or more attaching and / or tethering sites operable to attach and / or tether said object or a further object secured to said object.

8. The sensing device as claimed in any preceding claim, wherein the device comprises an integrated circuit and wherein the two or more driving elements and the at least one sensing element comprises electrodes integrated into the integrated circuit,9. The sensing device of any preceding claim, wherein the two or more driving elements comprise a first driving element and a second driving element and wherein the difference in impedance comprises a difference between a first impedance between the first driving element and the at least one sensing element and a second impedance between the second driving element and the at least one sensing element.

10. The sensing device as claimed in any preceding claim, wherein the sensed response comprises a combination of sensed responses from each of the two or more signals, wherein the difference in their phase and / or other electrical property of the two or more signals is such that the sensed responses substantially cancel to allow a contribution from the rotational motion of the objects in said fluid to be obtainable from the sensed response signal.

11. The device as claimed in any preceding claim comprising processing circuitry configured to process the sensed electrical response to determine the rotational property and / or at least further property associated with the object.

12. The device as claimed in any preceding claim wherein the rotational property and / or at least one further property comprises a rotational property of the object or an attached rotary motor or a further property of the fluid based on the rotational property of the object or attached rotary motor.

13. The device as claimed in any preceding claim, wherein the two or more respective signals have a signal frequency and / or a range of signal frequencies and wherein the sensed response signal is dependent on the signal frequency.

14. The device as claimed in any preceding claim, wherein the driving circuitry is configured to drive the driving elements to produce a pair of differential and / or complimentary signals.

15. The device as claimed in any preceding claim, wherein the two or more respective signals, optionally a pair of the two or more signals, have a phase difference between 135 and 180 degrees, optionally wherein the pair of signals are substantially out of phase.

16. The device as claimed in any preceding claim, wherein the two or more respective signals are driven at a signal frequency in the range of 100 kHz to 100 MHz and / or wherein the two or more respective signals are oscillating and / or alternating current signals.

17. The device as claimed in any preceding claim, wherein the two or more driving elements and the at least one sensing element are arranged in a triplet configuration comprising a sensing element provided between a first and a second driving element, optionally wherein the first and second driving elements are provided at an equal distance to the at least one sensing element.

18. The device of any preceding claim, wherein a plurality of groupings of driving elements and sensing elements are distributed across one or more surfaces, wherein the groupings are characterised by a spacing between driving and sensing elements and / or a relative orientation of the driving and / or sensing elements, and wherein at least one of the spacing and / or relative orientation is different between two or more groupings.

19. The device of any preceding claim, wherein the spacing between each driving element and the at least one sensing element is in the range between 0.1 pm to 50 pm, optionally 0.5 to 10 pm and / or wherein the sensing and / or driving element have at least one dimension in the range 0.3 to 15 pm.

20. The device of any preceding claim, wherein the at least one sensing surface forms a part of a fluidic channel providing a fluidic path at or about, optionally past, the at least one sensing element.21 . The device of claim 20, when dependent on claim 8, wherein the fluidic channel has a size corresponding to size of the integrated circuit22. The device of any preceding claim, wherein the object has at least one dimension, for example, a height, length or width, in the range 10 nm to 10 urn.

23. The device of any preceding claim, wherein the object is rotated or otherwise moved by a synthetic or natural nanometre or micron-sized rotary motor, optionally, wherein the rotary motor comprises at least one of: a bacterial flagellar motor; a F1 , Fo, Fo-ATP synthase, or equivalents for the Vacuolar-type ATPase (V-ATPase); a birefringent particle24. The device of any preceding claim, wherein the at least one further property comprises at least one of: a) an electrochemical gradient of a cell; b) a property related to flow and / or movement of the fluid, for example, a flow rate and / or viscous load; c) a chemical property of the fluid, for example, an identity of a molecule or chemical element in the fluid.

25. A method of sensing a rotational property and / or a further property associated with one or more objects, for example, nanometre or micron sized objects in a fluid, comprising: driving two or more driving element using two or more respective signals characterised by a difference in at least their phase and / or other electrical property;sensing a response signal in response to driving the driving element, wherein the difference in at least the phase and / or other electrical property of the two or more respective signals causes the sensed response signal to be dependent on at least a difference in impedance between each of the two or more driving elements and the at least one sensing element such that a rotational property of one or more objects in the fluid and / or at least one further property for the one or more objects or for the fluid is obtainable from the sensed response signal, wherein the rotational property and / or at least one further property comprises a speed of rotation and / or a frequency of rotation and / or a direction or rotation and / or a change in direction of rotation.

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