Patent Description:
The present invention is in the field of motion detector adapted to detect micro- or nanometer motion of small scale objects.

Techniques are available to detect cells and bacteria using micro- and nanosystems. These are however of limited use for biology, as they often destroy the live specimen, typically by requiring a vacuum environment. This limitation may pose a problem for advancement in further study and advancement in biology since it is not possible to look into processes that occur in live specimens, such as a metabolism thereof, growth thereof, and self-assembly and response to external stimuli or drugs.

These motion detectors are typically provided with an oscillator. Recently detectors have been developed having a flexible sample support in the form of a cantilever, or an optical fiber, or a piezoelectric system, capable of fluctuating, such as in <CIT>. The displacement of the cantilever, typically flexing thereof, can be measured quite accurately using e.g. an optical system, typically comprising a mirror a laser, and photodiodes, which is capable of measuring a deflection of the cantilever. Movement detection is limited to a nanoscale or larger scale motion. The same author, <NPL>) recites the use of an innovative nanoscale motion sensor in life-searching experiments in Earth-bound and interplanetary missions. The technique exploits the sensitivity of nanomechanical oscillators to transduce the small fluctuations that characterize living systems. The intensity of such movements is an indication of the viability of living specimens and conveys information related to their metabolic activity. The nanomotion detector can assess the viability of a vast range of biological specimens and that it could be the perfect complement to conventional chemical life-detection assays. Indeed, by combining chemical and dynamical measurements an unprecedented depth in the characterization of life in extreme and extraterrestrial environment may be achieved. <NPL>) recite that atomic layer crystals are emerging building blocks for enabling new two-dimensional (2D) nanomechanical systems, whose motions can be coupled to other attractive physical properties in such 2D systems. Optical interferometry has been very effective in reading out the infinitesimal motions of these 2D structures and spatially resolving different modes. To quantitatively understand the detection efficiency and its dependence on the device parameters and interferometric conditions, they present a systematic study of the intrinsic motion responsivity in 2D nanomechanical systems using a Fresnel- law-based model. They find that in monolayer to <NUM>-layer structures, MoS2 offers the highest responsivity among graphene, h-BN, and MoS2 devices and for the three commonly used visible laser wavelengths (<NUM>, <NUM>, and <NUM>). Their results elucidate and graphically visualize the dependence of motion transduction responsivity upon 2D material type and number of layers, vacuum gap, oxide thickness, and detecting wavelength, thus providing design guidelines for constructing 2D nanomechanical systems with optimal optical motion readout.

So the prior art detectors and sensors, for certain applications, are not sensitive enough. Typically they can not detect motion of a smaller living specimen, such as a single live bacterium or a virus. For certain application also a faster response is required such as revealing the status of the living organism in few seconds after a drug susceptibility test. Sometimes cost, size, multiplication, and complexity may be a challenge as well, in addition.

The present invention therefore relates to an improved two-dimensional (2D) motion detector, which solves one or more of the above problems and drawbacks of the prior art, providing reliable results, without jeopardizing functionality and advantages.

The invention is defined in the claims. It is an object of the invention to overcome one or more limitations of the prior art and provides a motion detector that is far more sensitive. The invention relates inter alia to a detector as defined in the claims, which can be used as a sensor, such as for metabolic activity of a sample, such as a single cell, a single bacterium, a single virus, or even a single biomolecule, by recording motion thereof. The detector comprises a suspended 2D material, e.g. a single layer of graphene, of MoS<NUM>, of hexagonal-BN, or any other atomically thin material, which may be a heterogeneous material. On the suspended material layer said single living matter may be provided, at micro- or nanoscale attached to its surface or in close proximity of its surface. The measurement means may be one from the group comprising a Fabry-Perot interferometer, a Michelson or an optical interferometer, a laser Doppler vibrometer, or one or more capacitor electrodes cooperating with the 2D material as the opposite electrode of the capacitor, or an imaging system for measuring the position of the living matter on the suspended material. In other implementations the resistance of the 2D material (which can be chosen to be piezoresistive) is used to monitor activities of the living matter on top of the 2D material. The to be measured actuation of the sample is induced by the activity or fluctuations of the above single sample object of interest, changing physical/chemical properties of said suspended 2D material. The suspended material is carrying the living matter and typically also a solid, liquid, or viscous fluid that contains a controllable concentration of its favourable growth environment. The present detector may be used in methods to supply medicine, toxins, or radiation to the living matter and study the effect on their activity are described. It is found that motion of the said 2D material is directly related to the activity of the sample, such as the single living organism, that can in turn be used for, among others, drug susceptibility testing. This present detector may be used as stand-alone technology, that e.g. can make drug susceptibility testing available at point of care within seconds. It is particularly suited for life science and health applications.

The present detector is validated in laboratory tests typically using E. coli strains. In an example the detector was able to distinguish between living bacterium in a blank set-up, living bacterium being resistant to added antibiotic, and bacterium under stress (dying) being susceptible to added antibiotic (see below).

The below are considered to relate to distinguishing features over the prior art, and effects thereof.

Applications in healthcare centres, ranging from general practitioners to hospitals, are envisaged, as well as applications in biology, physics, and medical labs. As the invention is sensitive at the single cell level, only very small amounts of living matter are needed for its operation. For example, sample preparation times does not need multiplication (cell division) and cell culturing before making measurements. This enables use at the point of care (general practitioners) with analysis times in the order of <NUM> minutes. It enables precision medicine: e.g. a bacterial sample from a patient is put on a chip that contains an array of different medicines. Motion of the bacteria on each part of the chip is monitored. The medicine that kills the bacteria (where motion is seen to decrease and stop in time) will be used on the patient.

So the present detector provides a way to probe the motion of a small sample, such as a single live organism, while being alive, without intervening in their natural physiology or behaviour. The technology is simple and cost efficient, such as a point-of-care testing technique for drug susceptibility, for preventing overuse of drugs, and for testing bacterial resistance to antibiotics. The inert and carbonous nature of graphene makes it ideally suitable for combining with organic living materials. The impermeability of the suspended graphene helps to make sure that the cavity below the said suspended material remains filled with air, such that the mechanical stiffness of the said suspended material stays low and sensitive to motion of the living matter. The mechanical strength of 2D materials like graphene ensures that the living material doesn't tear the material apart, despite its thin nature.

The present sensor assembly is dedicated for activity monitoring of a living microorganism or living nano-organism. The assembly is equally applicable to monitoring of other movement on nanoscale or picoscale. The sensor assembly comprises a 2D microscale motion detector, which is referred to as "microscale" as in plane dimensions thereof are in the low microscale, whereas a thickness typically is in the low nanoscale or even below a nm, the motion detector adapted to act as a sample receiver, such as for receiving the living organism, which organism may be provided in a liquid, such as in a droplet, comprising an inert suspended layer, onto which the living organism may be provided, and in view of the organism and optional liquid the layer is chemically and biologically inert, wherein the suspended layer is <NUM>-<NUM> atoms thick, at least one support for the suspended layer, which support may extend over the full boundary of the suspended layer, or may be provided at a part of the suspended layer, such as at two or more opposite edges, and a read-out system adapted for measuring alteration of the suspended layer. Therewith movement of e.g. the living organism over extremely small distances, or even of movement causing a centre of mass of the microorganism to shift, which may be in the order of picometers, can be measured accurately and reproducibly.

In a second aspect the present invention relates to a chip comprising at least one 2D microscale motion detector according to the invention. The present 2D microscale motion detector can easily be integrated into a chip, or any other micro-electronic structure, and can be integrated by using typically used semiconductor technology. Therein electrical connections, controls, and even micro-fluidic elements can be introduced simply. So the present invention also relates to an electronic device comprising a sensor assembly or chip according to the invention, and at least two channels each individually in electrical connection with the read-out system, such as <NUM>-<NUM> channels, at least one readout line.

In a third aspect the present invention relates to a method for operating the sensor assembly according to the invention, comprising providing a volume of liquid, the volume being < <NUM>µl, preferably < <NUM>µl, such as < <NUM>µl, the volume comprising a microorganism, or living cell constituent, or virus, and measuring motion of the microorganism, or living cell constituent, or virus, over time. Living cell constituents may relate to DNA, to RNA, to proteins, to enzymes, and so on, and fragments thereof, and combinations thereof. Also more than one organism, or cell constituent, or virus, can be measured accordingly. Examples of such measurements are given below.

In a fourth aspect the present invention relates to a disposable sample stage comprising a 2D microscale motion detector adapted to act as a sample receiver, for receiving a volume of liquid, the volume being < <NUM>µl, comprising an inert suspended layer, wherein the suspended layer is <NUM>-<NUM> atoms thick, and at least one support for the suspended layer, and typically also a substrate on which the support may be provided.

Advantages of the present description are detailed throughout the description.

In an exemplary embodiment of the present sensor assembly material of the suspended layer may be a two-dimensional crystal providing interlayer van der Waals interactions in a direction perpendicular to the layer surface, and is preferably selected from graphene, hexagonal-BN, black phosphorus, transition metal dichaclogenides, wherein the metal is preferably selected from Mo, W, Nb, and wherein the chalcogen is preferably selected from S, Se and Te, such as MoS<NUM>, NbSe<NUM>, and WSe<NUM>, and combinations thereof.

In an exemplary embodiment of the present sensor assembly the read-out system may be selected from a Fabry-Perot interferometer, a Michelson interferometer, an optical interferometer, a laser Doppler vibrometer, one or more capacitor electrodes, a piezoelectrical element, a piezoresistive element, an impedance analyser, and combinations thereof.

In an exemplary embodiment of the present sensor assembly alteration of the suspended layer changes at least one physical characteristics thereof selected from deflection, resonance frequency, reflection spectrum, transmission spectrum, optical adsorption, orientation of at least part of the suspended layer, optical interference, 2D crystal structure, electromagnetic properties, such as resistivity, conductivity, and combinations thereof.

In an exemplary embodiment of the present sensor assembly the read-out system may comprise a laser for providing light, an optical system for directing light from the laser to the sample, an optical system for directing reflected light from the sample to a photo detector, such as a photo diode, optionally an amplifier for amplifying detected light response, and a recorder for representing motion, such as an oscilloscope.

In an exemplary embodiment of the present sensor assembly the suspended layer may be <NUM>-<NUM> atoms thick, such as <NUM> atoms thick. It is found that a response for a thinner layer is better.

In an exemplary embodiment of the present sensor assembly the suspended layer may be <NUM>-<NUM> wide, such as <NUM>-<NUM> wide.

In an exemplary embodiment of the present sensor assembly the suspended layer may be <NUM>-<NUM> broad, such as <NUM>-<NUM> broad.

In an exemplary embodiment of the present sensor assembly the suspended layer may have a stiffness of <<NUM> N/m, preferably < <NUM> N/m, more preferably < <NUM> N/m, even more preferably <<NUM> N/m, such as < <NUM> N/m, and typically <<NUM> N/m. The stiffness can be measured using an AFM or STM, applying a force (N) to the layer, and measuring the (vertical) displacement of the layer, at the position of the force.

In an exemplary embodiment of the present sensor assembly the suspended layer may have a Youngs modulus of ><NUM> GPa, such as ><NUM> GPa (ASTM E1111).

In an exemplary embodiment of the present sensor assembly the suspended layer may have a weight of <<NUM>-<NUM> kg, preferably <<NUM>-<NUM> kg, such as <<NUM>-<NUM> kg. The very small weight has amongst others as advantage that very small movements, of otherwise also very light objects, such as microorganism, can be measured very accurately.

In an exemplary embodiment of the present sensor assembly under the suspended layer a cavity of > <NUM> height is provided, such as > <NUM>, such as <NUM>. Such sub-micrometre cavities can be provided using semi-conductor technology, such as by using a mask, and wet- or dry-etching. Very well dimensioned cavities can be provided thereby. The cavity may be fully surrounded by the substrate, the at least one support, and the suspended layer, or partly surrounded thereby, such as for <NUM>-<NUM>% of its boundary area.

In an exemplary embodiment of the present sensor assembly the cavity may be filled with a fluid, such as a gas or liquid. The gas may be an inert gas, such as nitrogen or a noble gas, whereas the liquid may be water, or a physiologically acceptable fluid.

In an exemplary embodiment of the present sensor assembly the at least one support may comprise an electrically insulating material, such as with an electrical conductivity σ (<NUM>) of <<NUM>-<NUM> S/m, preferably <<NUM>-<NUM> S/m, such as silicon oxide, silicon nitride, and silicon carbide. Therewith electrical connections and the like can be provided, being in contact with the detector, and further being insulated.

In an exemplary embodiment of the present sensor assembly the at least one support may have a height of <NUM>-<NUM>, preferably <NUM>-<NUM>, such as <NUM>-<NUM>.

In an exemplary embodiment of the present sensor assembly the at least one support may be provided on a substrate, such as a silicon substrate.

In an exemplary embodiment of the present sensor assembly the suspended layer, the at least one support, and substrate, are each individually non-toxic, and at least partly support organism activity, such as support appropriate cellular activity, including the facilitation of molecular and mechanical signalling systems, such as in order to optimise tissue regeneration, without eliciting any undesirable effects in those organisms, or inducing any undesirable local or systemic responses in the eventual host.

In an exemplary embodiment the present sensor assembly may further comprise a humidity chamber for receiving the suspended layer and a sample. Therewith controlled experiments can be performed, in a for the organism at least partly favourable environment.

In an exemplary embodiment the present sensor assembly may comprise an array of sample receivers, therewith providing the opportunity to perform a series of parallel and/or sequential experiments.

In an exemplary embodiment the present sensor assembly may comprise a chip, such as mentioned above.

In an exemplary embodiment of the present method a chemical may be added, wherein the chemical is preferably selected from pharmaceuticals or potential pharmaceuticals, such as anti-biotics, such as kanamycin, and chloramphenicol, and measuring a response of the microorganism, or living cell constituent, or virus, to the chemical over time. Examples thereof, and the response measured, are given below.

In an exemplary embodiment of the present method the liquid may comprise at least one of nutrition for the microorganism or for the living cell constituent or for the virus, a physiological liquid, and a metabolic support compound.

The invention will hereafter be further elucidated through the following examples which are exemplary and explanatory of nature and are not intended to be considered limiting of the invention. To the person skilled in the art it may be clear that many variants, being obvious or not, may be conceivable falling within the scope of protection, defined by the present claims.

The figures are detailed throughout the description, and specifically in the experimental section below.

<FIG> shows the sensor assembly <NUM> comprising a sample receiver <NUM> with inert 2D material layer <NUM> acting as the motion detector. The 2D material is suspended over a cavity <NUM> using at least one support <NUM>. Such geometry can be obtained using semiconductor technology, such as by using a mask, and wet- or dry-etching.

Turning back to <FIG>, it is shown that the sensor assembly also comprises a chip <NUM> that sits on a substrate <NUM>. The substrate <NUM> may comprise an array of sample receivers <NUM> on top of an electrical device <NUM>.

In the embodiment of <FIG> also a liquid droplet <NUM> is dispensed on top of the sample receiver <NUM>. The droplet contains micro-organisms <NUM> and nutrition <NUM>. Chemicals <NUM> can be added to the droplet to change the behaviour of the micro-organism. The micro-organism can be also adhered to the 2D material and its motion can be probed by a read-out system <NUM>.

<FIG> shows a read-out system that can be used for monitoring the metabolic activity of micro-organisms. In the embodiment of <FIG> a red helium-neon laser <NUM> is directed through optical components <NUM> on the 2D material layer <NUM> which is placed in a controlled humidity chamber <NUM>. The intensity of the reflected light from the chip <NUM> is altered by the motion of the micro-organism <NUM> in the liquid droplet <NUM> that in turn moves the suspended 2D material <NUM>. This intensity is then measured by a photodiode <NUM> connected to an oscilloscope <NUM>.

In one example a liquid droplet <NUM> containing micro-organism E. coli bacteria <NUM> and nutrition Lysogeny broth solution <NUM> has been dispensed on the sample receiver <NUM> comprising an array of single layer chemical vapour deposited graphene as the inert suspended layer <NUM>. The motion is read out using the measurement system described in <FIG>. The motion of the suspended layer is traced in a timeframe of a few seconds in the presence and absence of chemicals <NUM> and micro-organisms <NUM>.

<FIG> shows the motion of the 2D material in the presence of the droplet <NUM> mixed with only the nutrition <NUM>. This trace shows almost no fluctuations, indicating the absence of bacteria.

<FIG> shows the motion after adding bacteria <NUM>. This trace shows large fluctuations associated with the metabolic activity of the bacteria.

<FIG> shows the motion after addition of Chloramphenicol antibiotic <NUM> that kills the bacteria. No fluctuations are observed as a result of no bacterial metabolic activity.

<FIG> Shows the variance of the time traces given in <FIG>. It can be observed that the variance drops about three times after adding antibiotic to the droplet.

In another example a liquid droplet <NUM> containing micro-organism E. coli bacteria <NUM> and nutrition Lysogeny broth solution <NUM> has been dispensed on the sample receiver <NUM> comprising an array of silanized natural crystal exfoliated <NUM> few layer thick graphene as the suspended material <NUM>. <FIG> show the time traces of the suspended layer in a timeframe of twelve minutes.

<FIG> shows the motion of the suspended layer with adhered bacteria <NUM>. This time trace shows large fluctuations associated with the metabolic activity of the bacteria.

<FIG> shows the motion after addition of Kanamycin antibiotic <NUM> to which the micro-organism is resistant. No change in the fluctuations is observed as a result antibiotic resistance.

<FIG> shows the motion after addition of Chloramphenicol (CM) antibiotic <NUM> that kills the bacteria. No fluctuations are observed as a result of no metabolic activity of the bacteria. <FIG> Shows the variance of the time traces given in <FIG>. It can be observed that the variance drops about hundred times after adding Chloramphenicol to the droplet. However, almost no change in the variance is observed after adding Kanamycin (Ka).

<FIG> show the amplitude spectra of the time traces associated with <FIG>. A tenfold decrease is observed in the average amplitude of the spectrum after adding Chloramphenicol antibiotic to the droplet containing E. coli bacteria.

Claim 1:
Sensor assembly (<NUM>) for activity monitoring of a living microorganism or living nano-organism comprising
a sample receiver (<NUM>) for receiving a volume of liquid, the volume being < <NUM>µl, comprising a 2D microscale motion detector (<NUM>),
the motion detector comprising an inert suspended layer (<NUM>), wherein the suspended layer is <NUM>-<NUM> atoms thick, at least one support (<NUM>) for the suspended layer, and
a read-out system (<NUM>) configured for measuring alteration of the suspended layer.