A method of determining a state of a subject

Abstract

A method of determining a state of a subject is provided The method may comprise applying a liquid composition comprising a thermo-responsive polymer solvated therein to a surface of the subject, contacting a sensor with the liquid composition, and drying the liquid composition to form a conformal thermo-responsive polymer layer, thereby affixing the sensor to the surface of the subject, wherein the conformal thermo-responsive polymer layer is operable as a capacitive coupling layer to allow capacitive acquisition of a signal from the subject by the sensor for determining the state of the subject. A kit for preparing a device for determining a state of a subject, and use of the method and kit are also provided.

Description

A METHOD OF DETERMINING A STATE OF A SUBJECTCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of priority of Singapore patent application no.1020250050 IX, filed 25 February 2025, the contents of which being hereby incorporated by reference in its entirety for all purposes.TECHNICAL FIELD

[0002] Various embodiments relate to a method of determining a state of a subject, a kit for preparing a device for determining a state of a subject, and use of the method and kit.BACKGROUND

[0003] State of the art sensors for determining a state, such as general health and well-being, of a subject, may generally be classified into non-invasivc sensors and invasive sensors. Such sensors may be used, for example, in potential measurement for human and plant electrophysiology.

[0004] Non-invasive sensors may involve use of wet electrolyte or hydrogel as the interface between conductors and object surfaces, or dry sensors in the form of pins, sheets or thin films. Invasive sensors, on the other hand, may involve piercing or implanting into an object under study for measurements.

[0005] Ideally, the sensors should be able to maintain stable recording for a long time with good attachment, and should not cause damage to the monitored surface.

[0006] hi reality, however, non-invasive electrodes used for both human and plant bio-signals are unable to achieve long-term attachment with concurrent high surface conformability. For example, non-invasive sensors that use wet electrolyte or hydrogel as the bridging material orinterface between conductors and object surfaces tend to dry out within hours, resulting in instability for long-term monitoring. Therefore, although such sensors may have high conformability, they suffer from dehydration and are generally limited to short-term applications of within a day. Dry sensors, on the other hand, face difficulty in maintaining high conformability with rough surfaces. Dry conductive electrodes with long-term capabilities are rigid, which limits sensor attachment to flat surfaces.

[0007] Invasive sensors, which are generally preferred in commercial settings, result in additional damage to the object under monitoring, which is undesirable for plants and animals including humans, and can induce noises in the signals acquired. Invasive electrodes used for long-term measurements on plants cause mechanical damage to monitored surface.

[0008] Tn light of the above, there remains a need for an improved method of determining a state of a subject that overcomes or at least alleviates one or more of the above-mentioned problems.SUMMARY

[0009] Tn a first aspect, a method of determining a state of a subject is provided. The method may comprise applying a liquid composition comprising a thermo-responsive polymer solvated therein to a surface of the subject, contacting a sensor with the liquid composition, and drying the liquid composition to form a conformal thermo-responsive polymer layer, thereby affixing the sensor to the surface of the subject, wherein the conformal thermo-responsive polymer layer is operable as a capacitive coupling layer to allow capacitive acquisition of a signal from the subject by the sensor for determining the state of the subject.

[0010] hi a second aspect, a kit for preparing a device for determining a state of a subject is provided. The kit may comprise a liquid composition comprising a thermo-responsive polymer solvated therein, wherein in use, the liquid composition forms a conformal thermoresponsive polymer layer on a surface of the subject upon disposing of the liquid composition on the surface of the subject with subsequent drying, wherein the conformal thermo-responsive polymer layer is operable as a capacitive coupling layer to allow capacitive acquisition of a signal from the subject for determining the state of the subject, and a sensor for affixing to the surface of the subject via the conformal thermo-responsive polymer layer.

[0011] Tn a third aspect, use of a method according to the first aspect or a kit according to the second aspect to obtain one or more readings of electrical potential difference and / or electrical impedance as a signal for determining a state of a subject over an extended time period of one month or more is provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In the drawings, like reference characters generally refer to the same parts throughout the different views. The drawings are not necessarily drawn to scale, emphasis instead generally being placed upon illustrating the principles of various embodiments. In the following description, various embodiments of the invention are described with reference to the following drawings.

[0013] FIG. 1 is a schematic diagram showing application process of a thermo-responsive polymer-interfaced sensor disclosed herein on an object surface, and final state of a dry thermo-responsive polymer sensor as disclosed herein.

[0014] FIG. 2A shows optical images of a hole 242 created by an invasive metal electrode on a plant when fresh i.e. when the hole was newly created, as compared to the same hole 30 days later. The hole 242 is depicted by the dashed line in the figure. Dimensions of the hole 242 increased which demonstrates mechanical instability of invasive electrodes on plants. Scale bar denotes 500 pm.

[0015] FIG. 2B is a graph showing length (pm) and width (pm) of the hole shown in FIG.2A, at fresh 250, i.e. when the hole was newly created, as compared to the same hole 30 days later 252. Dimensions of the hole increased, which demonstrates mechanical instability of invasive electrodes on plants.

[0016] FIG. 3A shows scanning electron microscopy images of a silver / silver chloride (Ag / AgCl) electrode before and after insertion into a plant, depicting chemical instability of invasive electrodes on plants, for pristine plant and the same plant 30 days after an invasive electrode was inserted. Scale bar denotes 5 pm.

[0017] FIG. 3B is a graph showing atomic % of silver (Ag), chlorine (Cl) and oxygen (O) for pristine plant 350 and plant with invasive electrode inserted 352. Results show chemical instability of invasive electrodes on plants.

[0018] FIG. 4 shows stability comparison between invasive electrode 450 and dry thermo-responsive polymer 452. The invasive electrode 450 shows obvious signal drift over time while the dry thcrmo-rcsponsivc polymer 452 shows high stability of signals over time.

[0019] FIG. 5 shows stability comparison between fresh agar 550 and dry thermo-responsive polymer 552.

[0020] FIG. 6 shows signal-to-noise ratio of fresh agar 650 as compared to dry thermo-responsive polymer 652 over a month of application.

[0021] FIG. 7 shows signal acquisition after 50 days of application on the peperomia plant for fresh agar 750 as compared to dry thermo-responsive polymer 752, at 0, 5, 10, 15, 20, 25, and 30 days.

[0022] FIG. 8 A shows chlorophyll content (SPAD) of leaves without coverage of the dry thermo-responsive polymer sensor 850, and with coverage of the dry thermo-responsive polymer sensor 852 for 1 month, at 0, 5, 10, 15, 20, 25, and 30 days.

[0023] FIG. 8B is a graph showing open stomata percentage of leaves with coverage of the dry thermo-responsive polymer sensor (“El ectrode- covered”), and without coverage of the dry thermo-responsive polymer sensor (“Bare leaf’) for 1 month.

[0024] FIG. 9 is a graph of plant electrical signals acquired and shows all conductors of silver / silver chloride with thermoresponsive polymer prepared in potassium chloride solution (Ag / AgCl-KCl, 950), carbon nanotube with thermoresponsive polymer prepared in potassium chloride solution (CNT-KC1, 952), gold with thermoresponsive polymer prepared in potassium chloride solution (Au-KCl, 954), silver / silver chloride with thermoresponsive polymer prepared in deionized water (Ag / AgCl-DI, 956), carbon nanotube with thermoresponsive polymer prepared in deionized water (CNT-DI, 958), and gold with thermoresponsive polymer prepared in deionized water (Au-DT, 960), are able to record plant electrical signals under flame stimulus.

[0025] FIG. 10 is a graph of potential (V) against time (s) from 0 to 3000 s showing the plant electrical signals acquired for 3 plants. Each plant has dehydrated thcrmogclling polymers of PEG / PPG / PCL (“10 % EPC”, 1058) and PEG / PPG with phenyl functionalization (“5% PET”, 1054) with conductor and all electrodes are able to record plant electrical signals under flame stimulus.

[0026] FIG. 11 shows the plant electrical signal acquired for invasive metal placed at stem 1150 and dry thermo-responsive polymer electrode placed on leaf 1152, and that application of invasive electrodes on stems is unable to obtain light information about plants, which is in contrast with the light peak observed using the dry thermo-responsive polymer electrode on leaf whereby the light information is obtained.

[0027] FIG. 12 shows a photograph of an invasive electrode on leaf which produces a hole, causing mechanical damage to the plants and that application of invasive electrodes on softleaves results in holes accompanied with large signal drift as shown by 1250. This is in contrast with signal obtained from dry thermo-responsive polymer 1252.

[0028] FIG. 13 shows a dry carbon nanotube conductive electrode placed on top of the hairy plant surface and that dry conductive electrodes have poor conformability on hairy surface with strong mechanical disturbance under touch and wind as shown by signal 1350, whereas dry thermo-responsive polymer is able to maintain high signal quality under mechanical disturbance as shown by 1352. Scale bar denotes 1mm.f0029] FIG. 14A shows an example of plant electrophysiology monitoring, for long term measurement of plant electrical signals over a month, whereby 1454 denotes signals from control electrodes and 1458 denotes signals from plant electrodes. The image shows the ability to measure month-long signals under varying light / dark cycles applied to plants.

[0030] FIG. 14B shows an example of plant electrophysiology monitoring, with accurate measurement on plant circadian cycles when light / dark duration was changed. 1454 denotes signals from control electrodes and 1458 denotes signals from plant electrodes.DESCRIPTION

[0031] The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practised. These embodiments are described in sufficient detail to enable those skilled in the art to practise the invention. Other embodiments may be utilized and structural, logical, and electrical changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0032] Various embodiments refer in a first aspect to a method of determining a state of a subject. The state of a subject may refer to general health and well-being, such as healthconditions and growth trends, of a subject in the form of living things such as animals, humans, and plants. Parameters or signals may be collected from a subject for determining a state of the subject. Information on whether the subject is thriving or requires intervention may be derived from the collected signals.

[0033] For example, onset of stress that a living thing is experiencing may be detected by the collected signals. An example may include measuring electrical potential difference between two points on plants in plant electrophysiology, whereby an obvious change in potential difference may be recorded when plants are wounded.

[0034] Methods disclosed herein may involve use of a dry thermo-responsive polymer as an interface between a conductor and a surface of interest to provide a non-invasive sensor, which is conformal to rough surfaces with long-term stability. The sensor provided by methods disclosed herein may be wearable, and may advantageously be used for long-term continuous measurement of parameters, such as electrical potential differences, to allow determining of a state of a subject. Methods disclosed herein may provide for non-invasive, long-term and stable electrical signal measurement on biological surfaces.

[0035] As mentioned above, methods disclosed herein may involve use of a dry thermo-responsive polymer as an interface between a conductor and a surface of interest. The term “dry” may refer to moisture (or water) content of a thermo-responsive polymer layer when the thermo-responsive polymer layer dehydrates to a stable moisture (or water) content in the environment of intended usage. For example, a plant growth environment in exemplified embodiments was at about 25 °C and about 60 % relative humidity, and water content of the “dry” thermo-responsive polymer was below 5 wt%. It was found that the water content stabilizes or has minimal change after air drying on plants for 1 day.

[0036] Methods disclosed herein are distinguished from methods which focus on using a wet thermogel to interface with a subject such as plants to achieve lower impedance, wherebyacquisition of signals using the wet thermogel is based on the ionic conductivity of the wet thermogel. Furthermore, signal acquisition with the wet thermogel may only be carried out for short-term measurements (up to 2 hours), since dehydration of the wet thermogel, which affects ionic conductivity measurements, may result otherwise.

[0037] The method disclosed herein may comprise applying a liquid composition comprising a thermo-responsive polymer solvated therein to a surface of the subject.

[0038] As used herein, the term “thermo-responsive” refers to materials that demonstrate altered physical characteristics at different temperatures. For example, the thermo-responsive polymer may respond to temperature, and be in a certain state, such as a solid state, above a certain temperature, and be in a different state, such as liquid state, at a lower temperature. The change in system morphology from a liquid-like sol into a solid-like gel may also be termed as “sol-gel transition”.

[0039] The thermo-responsive polymer may be any thermo-responsive polymer with suitable sol-gel transition properties. Generally, any thermo-responsive polymer with suitable sol-gel transition temperature above room temperature and with storage modulus at room temperature, preferably above 100 Pa, that allows strong and clean attachment of gel can be used.

[0040] The thermo-responsive polymer may, for example, comprise or consist of one or both a polyethylene glycol / polypropylene glycol / polycaprolactone urethane copolymer and a polyethylene glycol / polypropylene glycol / phenyl functionalized urethane copolymer.

[0041] In various embodiments, the thermo-responsive polymer is one or more of a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and polycaprolactone (PCL) and connected with urethane bonds, and a copolymer synthesized w'ith polyethylene glycol (PEG), polypropylene glycol (PPG) and l,5-hexadiene-3,4-diol and connected w'ith urethane bonds followed by thiol-ene click reaction with 2-phenylethanethiol.

[0042] In some embodiments, the thermo-responsive polymer comprises a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and polycaprolactone (PCL), connected with urethane bonds using hexamethylene diisocyanate (HMDI) as coupling reagent, and a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and l,5-hexadiene-3,4-diol and connected with urethane bonds using hexamethylene diisocyanate (HMD!) as coupling reagent followed by thiol-ene click reaction with 2-phenylethanethiol.

[0043] The thermo-responsive polymer may be at least substantially dissolved or be completely dissolved in a suitable solvent, for example, an aqueous medium such as water, to provide the liquid composition.

[0044] Applying the liquid composition to a surface of the subject may be carried out using any suitable method, such as, but not limited to, applying via a dropper, pipette, spraying, brush coating, or dip coating.

[0045] The method may comprise contacting a sensor with the liquid composition, and drying the liquid composition to form a conformal thermo-responsive polymer layer, thereby affixing the sensor to the surface of the subject.

[0046] In various embodiments, the sensor comprises an electrical conductor. Contacting the sensor with the liquid composition may comprise placing the electrical conductor in contact with the liquid composition. Contacting the sensor with the liquid composition may be carried out before drying of the liquid composition is carried out, so as to allow attaching of the sensor to the surface of the subject upon drying of the liquid composition.

[0047] The electrical conductor may be any suitable electrically conductive materials such as, but not limited to, metal and / or carbon-based materials.

[0048] In various embodiments, the electrical conductor may be one or more of silver / silver chloride, gold, carbon nanotube, and a composite formed from carbon nanotube and bacterial cellulose.

[0049] Drying the liquid composition may be carried out such that moisture content of the conformal thermo-responsive polymer layer remains essentially the same during the determining in an environment of intended usage.

[0050] As mentioned above, the term “dry” as used herein may refer to moisture (or water) content of a thermo-responsive polymer layer when the thermo -responsive polymer layer dehydrates to a stable moisture (or water) content in the environment of intended usage.

[0051] For example, a plant growth environment in exemplified embodiments was at about 25 °C and about 60 % relative humidity, and water content of the “dry” thermo-responsive polymer was below 5 wt%. It was found that the water content stabilizes or has minimal change after air drying on plants for 1 day.

[0052] Accordingly, methods disclosed herein may comprise drying the liquid composition under suitable conditions so that moisture content of the conformal thermo-responsive polymer layer remains essentially the same during the determining in an environment of intended usage.

[0053] For example, methods disclosed herein may comprise drying the liquid composition under suitable conditions so that moisture content of the conformal thermo-responsive polymer dehydrates to a stable water content, generally at 5 wt% or less, such as 4 wt% or less, 3 wt% or less, 2 wt% or less, or 1 wt% or less.

[0054] In various embodiments, drying the liquid composition is carried out such that moisture content of the conformal thermo -responsive polymer is 5 wt% or less.

[0055] The conformal thermo-responsive polymer layer may be operable as a capacitive coupling layer to allow capacitive acquisition of a signal from the subject by the sensor for determining the state of the subject.

[0056] The capacitive coupling may involve transfer of energy without any charge carriers. Advantageously, methods disclosed herein do not require ionic conductivity (such as that of gel), and may instead, use the dry state of a thermo-responsive polymer for long-term acquisition of signals (e.g., a month) for determining a state (such as general health and wellbeing) of a subject (such as an animal or a plant).

[0057] In applying a liquid composition comprising a thermo-responsive polymer solvated therein to a surface of the subject, whereby the surface may be a non-flat surface, such as a rough or hairy surface, with subsequent contacting of a sensor with the liquid composition, and drying the liquid composition to affix the sensor to the surface of the subject, a conformal thermo-responsive polymer layer may be formed on the non-flat surface of the subject, whereby contact with the subject is maximized. This may result in improved sensitivity for a method of determining a state of a subject as disclosed herein, whereby one or more readings (such as electrical potential difference as electrophysiological signals) of the subject obtained by the sensor may be enhanced.

[0058] By virtue of the thermo-responsive polymer layer being operable as a capacitive coupling layer to allow capacitive acquisition of a signal from the subject by the sensor for determining the state of the subject, methods disclosed herein do not depend on and are not carried out based on conductivity of the thermo -responsive polymer layer, and are therefore able to avoid issues relating to methods based on ionic conductivity (such as that of gel), whereby dehydration of gel-based electrodes may result in signal drift which is not suitable for long-term monitoring. The signal drift may be due to loss of conductivity as it reaches athreshold value for effective electrophysiological recording through ionic conductivity of the gel.

[0059] In various embodiments, the conformal thermo-responsive polymer layer is not ion conductive. As mentioned above, sensors based on methods disclosed herein do not depend on and are not canned out based on conductivity of the themo-responsive polymer layer, and are therefore able to avoid issues relating to methods based on ionic conductivity (such as that of gel), whereby dehydration of gel -based electrodes may result in signal drift which is not suitable for long-term monitoring.

[0060] In various embodiments, the method further comprises obtaining one or more readings of electrical potential difference and / or electrical impedance as the signal over an extended time period of one month or more.

[0061] Since the thermo-responsive polymer layer is stable in the dry state, it allows longterm monitoring, such as an extended time period of one month or more, 6 weeks or more, 2 months or more, or 10 weeks or more. Furthermore, w'hilc potential solutions to dehydration may, for example, include use of glycerol as a solvent in place of water, the glycerol may interfere with plant growth at high concentration, and have not shown long-term signal stability or compatibility on plants. Since methods disclosed herein do not require use of glycerol, such issues may advantageously be avoided.

[0062] The electrical potential difference may be measured as electrophysiological signals, for monitoring state of a subject, as a plant, over an extended time period, such as more than a month. The onset of stress that the subject is experiencing can be detected by the collected signals, as indication of whether the subject is thriving or requires intervention. For example, an obvious change in potential difference can be recorded when plants are wounded, to provide indication that intervention is required.

[0063] In a second aspect, a kit for preparing a device for determining a state of a subject is provided. The kit may be used for preparing a sensor for determining a state of a subject based on concepts disclosed herein.

[0064] The kit may comprise a liquid composition comprising a thermo-responsive polymer solvated therein, and a sensor.

[0065] In use, the liquid composition forms a conformal thermo-responsive polymer layer on a surface of the subject upon disposing of the liquid composition on the surface of the subject with subsequent drying, wherein the conformal thermo-responsive polymer layer is operable as a capacitive coupling layer to allow capacitive acquisition of a signal from the subject for determining the state of the subject.

[0066] The sensor comprised in the kit may be for affixing to the surface of the subject via the conformal thermo-responsive polymer layer.

[0067] Examples of suitable liquid composition, thermo-responsive polymer, and sensor have already been mentioned above.

[0068] As also mentioned above, any thermo-responsive polymer with suitable sol-gel transition temperature above room temperature and with storage modulus at room temperature, preferably above 100 Pa, that allows strong and clean attachment of gel can generally be used for application.

[0069] In various embodiments, the thermo-responsive polymer comprises or consists of one or both a polyethylene glycol / polypropylene glycol / polycaprolactone urethane copolymer and a polyethylene glycol / polypropylene glycol / phenyl functionalized urethane copolymer.

[0070] In some embodiments, the thermo-responsive polymer is one or more of a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and polycaprolactone (PCL) and connected with urethane bonds, and a copolymer synthesizedwith polyethylene glycol (PEG), polypropylene glycol (PPG) and l,5-hexadiene-3,4-diol and connected with urethane bonds followed by thiol-ene click reaction with 2-phenylethanethiol. roo7i] In specific embodiments, the thermo-responsive polymer comprises a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and polycaprolactone (PCL), connected with urethane bonds using hexamethylene diisocyanate (HMDI) as coupling reagent and a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and l,5-hexadiene-3,4-diol and connected with urethane bonds using hexamethylene diisocyanate (HMDI) as coupling reagent followed by thiol-ene click reaction with 2-phenylethanethiol.

[0072] The sensor may comprise an electrical conductor for contacting the conformal thermo-responsive polymer layer.

[0073] Examples of suitable electrical conductor have already been mentioned above. In various embodiments, the electrical conductor is one or more of silver / silver chloride, gold, carbon nanotubc, and a composite formed from carbon nanotubc and bacterial cellulose.

[0074] Various embodiments refer in a third aspect to use of a method according to the first aspect or a kit according to the second aspect to obtain one or more readings of electrical potential difference and / or electrical impedance as a signal for determining a state of a subject over an extended time period of one month or more.

[0075] As mentioned above, parameters or signals may be collected from a subject for determining a state of the subject. One or more readings of electrical potential difference and / or electrical impedance may be collected as a signal for determining a state of a subject over an extended time period of one month or more, and information on whether the subject is thriving or requires intervention may be derived from the collected signals.

[0076] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following non-limiting examples.EXAMPLES

[0077] As disclosed herein, a method of determining a state (such as general health and wellbeing) of a subject (such as an animal including human, or a plant) may be provided. The method may comprise• applying a liquid composition comprising a thermo-responsive polymer (such as poly(PEG / PPG / PCL urethane) (EPC)) solvated therein to a surface of the subject, • contacting a sensor (such as a wearable sensor) with the liquid composition, and • drying the liquid composition to affix the sensor to the surface of the subject.

[0078] Also disclosed herein is a kit for preparing a device for determining a state (such as general health and well-being) of a subject (such as an animal or a plant), the kit comprising • a liquid composition comprising a thermo-responsive polymer (such as poly(PEG / PPG / PCL urethane) (EPC)) solvated therein, wherein in use, the liquid composition forms a conformal thermo-responsive polymer layer on a surface of the subject upon disposing of the liquid composition on the surface of the subject with subsequent drying, wherein the conformal thermo-responsive polymer layer is operable as a capacitive coupling layer to allow capacitive acquisition of a signal from the subject for determining the state of the subject, and• a sensor (such as a wearable sensor) for affixing to the surface of the subject via the conformal thermo-responsive polymer layer.

[0079] Further disclosed herein is use of a method as presently disclosed or a kit as presently disclosed to obtain one or more readings of electrical potential difference and / or electricalimpedance as a signal for determining a state (such as general health and well-being) of a subject (such as an animal or a plant) over an extended time period of one month or more.

[0080] Examples disclosed herein provide a dry temperature -responsive or thermo-responsive polymer between a conductor and a surface of interest to achieve a non-invasive wearable sensor, which is conformal to rough surfaces with long-term stability. The dry thermo-responsive polymer-based wearable sensor may be conformally attached to the surface and be used for long-term continuous measurement of parameters such as electrical potential differences, and achieving non-invasive, long-term and stable electrical signal measurement on biological surfaces.

[0081] Ability of the sensor to conform to rough surfaces with long-term stability may result from use of a dry temperature-responsive or thermo-responsive polymer as an interlayer between a conductor and the recording object surface (e.g. human skin or plant) for long-term application. Use of sol-gel transition of thermo-responsive polymer allows formation of good and stable contact with the object surface in gel state. Such high conformability with the surface is retained in its dry state. This may provide for a non-invasive way of conformally attaching sensors on rough, uneven, and / or hairy surfaces (for example, human skin, plant leaves) for long-term monitoring of parameters that provide information about the state of the subject.

[0082] Any conductor can be used in embodiments disclosed herein, including but not limited to, silver / silver chloride (Ag / AgCl), gold (Au), and / or carbon nanotube (CNT). Deionised water (DI) and potassium chloride (KC1) were used to indicate whether the thermogelling polymer have ionic conductivity. All conductors were able to record plant electrical signals under flame stimulus. Presence or absence of ionic conductivity did not affect the measurements.

[0083] Any thermo-responsive polymer can be used in embodiments disclosed herein, including, but not limited to, PEG / PPG / PCL (EPC) and PEG / PPG / Phenyl functionalized thermogel (PET). Both dehydrated thermogelling polymers with conductor were able to record plant electrical signals under flame stimulus.

[0084] Recording object surface can be of different roughness and surface irregularities, such as that of a hairy plant surface.

[0085] I. Design concept of the sensor

[0086] A key issue which this technology addresses is concurrent conformability and stability. This may be carried out through use and treatment of a thermo-responsive polymer. In the experiments, the polymer was used in an aqueous solution, which maintained a solution state when kept at a low temperature and formed a gel when left at room temperature (known as sol-gel transition).

[0087] As illustrated in FIG. 1, to attach the sensor to an object, the thermo-responsive polymer solution 1031 (stored at low temperature) may first be applied on the object's surface 101 in a solution state, and the conductor 105 may then be applied on top of the polymer droplet 1031. This is shown in 1001.

[0088] When temperature increases, as shown by 1002, the polymer solution 1031 may transform into a semi-solid gel 1032 with stronger mechanical properties, which is able to secure the conductor 1032 without dropping easily. The solution state of the polymer 1031 helps to ensure conformal contact with both the conductor 105 and the object surface 101, with any uneven topography.

[0089] As the thermo-responsive polymer 1032 is able to maintain a gel state at room temperature, high conformability of the gel at the interface of both the object surface 101 and the conductor 105 may be maintained as the polymer gel undergoes dehydration.

[0090] Upon drying, as shown in 1003, the thermo-responsive polymer layer may form a thin dielectric 1033 that can acquire signals from the object 101 through capacitive coupling. Since drying of the polymer layer 1033 has taken place, further instability is not expected to take place during long-term usage.

[0091] II. Material preparation

[0092] Thermo-responsive polymer

[0093] Polymer used in the experiments was poly(PEG / PPG / PCL urethane), EPC in short. EPC is an amphiphilic copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and polycaprolactone (PCL), connected with urethane bonds using hexamethylene diisocyanate (HMDI) as the coupling reagent. EPC polymer synthesis protocols were as follows: EPC was synthesized from polyethylene glycol (PEG), polypropylene glycol (PPG), and poly(e-caprolactone)-diol (PCL-diol) using hexamethylene diisocyanate (HMDI) as a coupling reagent and dibutyltin dilaurate (DBTL) as a catalyst. HMDI was added in equal molar amount as the molar sum of PEG, PPG, and PCL-diol. Typically, 12 g of PEG (M„ = 2050 g mol’1, 5.85 xlO’3mol), 3 g of PPG (Mn= 2000 g mol’1, 1.50 x 10’3mol), and 0.15 g of PCL-diol (Mn= 2000 g mol’1, 7.50 x 10’5mol) were weighed out into a 250 mL round bottom flask. The reactants were then dissolved in 40 mL anhydrous toluene at 60 °C for 10 min before undergoing azeotropic distillation to remove moisture from the reactants. Azeotropic distillation was carried out until the reactants became a slurry. The slurry was dissolved in another 40 mL of anhydrous toluene at 60 °C for 10 min before doing a second round of azeotropic distillation. The slurry obtained was further dried under high vacuum for 3 h at 110 °C to ensure complete removal of water. After the high vacuum step was completed, the slurry was dissolved in 10 mL of anhydrous toluene and two drops of dibutyltin dilaurate (about 8 mg) were added sequentially. 1.23 mL (7.09 mmol, 103 %) of HMDI was then injected into the round bottom flask to start polymerization. The reaction wasdone at 110 °C for 24 h. During the first 4 h, 10 mL of anhydrous toluene was added each time whenever the reaction becomes too viscous to be stirred. After 24 h, the reaction solution was precipitated in 2 L of diethyl ether. The crude polymer product was blown dry and redissolved in 150 mL isopropyl alcohol (IPA) at 60 °C for 30 min.

[0094] Preparation of EPC solutions involved dissolving EPC polymer in a saline stock solution of desired concentration (10 mM potassium chloride (KC1)) and storing at 4 °C for 3 days for full solvation.

[0095] Conductor

[0096] The conductor used in the experiments was based on carbon nanotubes (CNT). They were 80 wt% CNT and 20 wt% bacterial cellulose (BC) composite. CNT powder (BuckyUSA, Bu-202) was first removed from impurities with immersion in 3M hydrochloric acid (HC1) solution for 12 hours. Filtration and washing of the CNT powder were conducted five times with de-ionised (DI) water. 64 mg of CNT and 26 mg of BC were dispersed in 0.2 mg / mL DI water and mixed using a blender. Vacuum filtration of the dispersion was conducted using a 9 cm filter membrane to obtain the thin CNT / BC film.

[0097] For connection with external testing systems, additional interconnects that bridge CNT / BC film and standard electrical wires were prepared. In detail, gold (Au) strips were prepared through thermal evaporation of 70 nm Au on flexible polyimide (PI) thin films (about 30 pm thickness). A 5 nm thick chromium (Cr) layer was first deposited to enhance adhesion between Au and PI. The coated thin films were cut into strips of 2 to 3 mm width for use. The CNT / BC film was cut into 3 mm x 3 mm pieces and pasted on the tip of Au strips using Ag conductive paint (Electrolube, SCP03B), followed by 60 °C drying in the oven.

[0098] III. Sensor performance benchmarking

[0099] Plant electrophysiology was used as an application case study to demonstrate the capability of this technology and showcase its advantages compared to state-of-the-art technologies.

[0100] Plant electrophysiological signals are the potential difference between two points on or within plants.

[0101] Current sensors for plant electrophysiological monitoring involve use of either invasive metal electrodes or noninvasive surface electrodes. For invasive electrodes, they result in additional damage to plant tissues and such damage may mask the detection of other signals. Furthermore, the mechanical instability of invasive electrodes, which can be seen from the noticeable hole enlargement after a 30-day insertion in plants (FIG. 2A and FIG.2B), can also cause signal instability.

[0102] In addition, invasive electrodes are unable to maintain chemical stability for long-term insertion in the complex chemical environment of plants (FIG. 3A and FIG. 3B).The mechanical and chemical instability of the invasive electrodes therefore results in a large drift in the acquired signals (FIG. 4).

[0103] Tn other words, invasive electrodes cause wounding to plants and are chemically unstable after long-term insertion. Application of invasive electrodes on stems is unable to obtain light information about plants. Application of invasive electrodes on soft leaves results in holes accompanied with large signal drift.

[0104] On the other hand, non-invasive electrodes are unable to achieve a conformable interface on plants with complex and hairy surfaces.

[0105] In contrast, technology disclosed herein can achieve a long-term conformable interface for stable monitoring of plant electrical signals. The current gold standard for noninvasive electrodes is the fresh agar electrodes. However, fresh agar is limited to working in the wet state and produces a large amount of drift within its working duration due todehydration (FIG. 5, 550). Such huge drift is absent in the technology disclosed herein (FIG.5, 552). Wet agar electrodes suffer from dehydration and can only be used for short term (less than 1 hour). During the dehydration, large signal drift was recorded. The dry thermo-responsive polymer electrode recorded a significant lower draft as compared to wet agar electrodes.

[0106] Besides drift, when the signal quality was benchmarked against fresh agar, the dry thermo-responsive polymer 652 achieved a comparable signal-to-noise ratio with fresh agar 650 over a month-long application (FIG. 6). It is important to note that in this comparison, fresh agar was used for each test within 2 hours, but technology disclosed herein was applied on day 0 and never reapplied for each test. The duration of monitoring can be much longer than 1 month, as the testing was restricted by the plant lifetime. When using plants of a longer lifetime, clear signals could still be acquired after 50 days of attachment (FIG. 7) These results show the superior signal quality and stability of the technology disclosed herein for long-term monitoring.

[0107] Moreover, the technology disclosed herein can achieve long-term compatibility with plants. A month-long attachment of the dry thermo-responsive polymer electrodes on plants showed minimal effect on their chlorophyll content and stomata opening / closing (FIG.8A and FIG. 8B), showcasing the good biocompatibility and safety of long-term application. Long-term application of dry thermo-responsive polymer has not shown any adverse effects on the plants. Chlorophyll content of bare leaves and leaves covered with dry thermo-responsive polymer were similar, without any distinct difference. Over a month of application, no difference in open stomata percentage for leaves with and without dry thermo-responsive polymer were recorded. These indicates that the application of the dry thermo-responsive electrode on leaf did not affect physiological activities of plants.

[0108] As can be seen from the above discussion, key technical features of the present technology may include

[0109] 1) Conformability to uneven surface and topography

[0110] 2) Stable long-term attachment on objects (more than a month)

[0111] 3) High-quality long-term potential signal measurements (more than a month)

[0112] 4) Long-term biocompatibility for application on plants (more than a month)

[0113] To the best of inventors’ knowledge, use of dry thermo-responsive polymer in electrical sensors has never been made public. Method of sensor attachment through sol-gel transition plus dehydration has never been made public. An electrical sensor that can conform to hairy surfaces while maintaining long-term stability is not known publicly.

[0114] Continuous recording of plant electrical signals for 31 days was achieved with the dry thermo-responsive polymer. There is no need for re-attachment or rehydration of electrode throughout the application duration.

[0115] Stable electrical signals of high signal-to-noisc ratio can be obtained. Signal maintains minimal drift as compared to conventional invasive and non-invasive wet electrodes, allowing measurement of true biosignals.

[0116] Advantageously, embodiments disclosed herein are able to address or at least alleviate problems relating to current technology relating to limitations of the application to objects that do not undergo extensive stretching or growth. This issue can be overcome by using dry temperature-responsive polymers that have viscoplastic properties and stretchable conductors.

[0117] Most of the materials used in the technology are synthesized / prepared by the inventors using methods developed by the inventors. To the best of the inventors’ knowledge, no such technology exists that can conform to rough surfaces while also performing stable long-term recording for months. Commonly used sensors for electrical signal acquisition areconductive dry electrodes or wet hydrogels. However, these electrodes are not capable of long-term stable recording on uneven topography. Other capacitive coupling methods commonly use dielectric cloth which requires clamping or additional adhesives on the object. Such electrodes have difficulties in achieving conformable attachment on uneven topography which might induce large noise in the signal acquisition process. Furthermore, clamping or the use of adhesion tapes might impose strains on the object and affect the quality and realness of signals acquired.rooii8i The present technology may be exploited as a universal application method for long-term, stable electrical signal monitoring on biological surfaces. Possible applications may include:

[0119] 1. Health monitoring of precious / delicate plant species in national reserves / conservatories / gardens / hotels, etc.

[0120] 2. Interactive interface between plants and humans for display, education, indoor decoration, wellbeing enhancement, etc.

[0121] 3. Health monitoring of crops in indoor farms

[0122] 4. Reporting / monitoring of environmental conditions (e.g., pollution, human presence and movement) using plants as living sensors, such as near factories including water treatment plants, oil refinement facilities, etc.

[0123] 5. Long-term and motion artefact-free electroencephalogram (EEG) monitoring on hairy scalp

[0124] 6. Long-term electrocardiogram (ECG) and electromyogram (EMG) monitoring on hairy skin

[0125] Further possible application of the technology may include, but are not limited to, health monitoring of crops indoors and in the field; health monitoring of precious / delicate plant species in national reserves / conservatories / gardens; reporting / monitoring ofenvironmental conditions (e.g., pollution, human presence and movement) using plants as living sensors, such as near factories including water treatment plants, and oil refinement facilities; interactive interface between ornamental plants and humans (e.g., touch-display, touch-sound) for display, education, indoor decoration, etc; long-term and motion artefact-free electroencephalogram (EEG) monitoring on hairy scalp; long-term electrocardiogram (ECG) and electromyogram (EMG) monitoring on hair}' skin.

[0126] Large-scale production of materials may be possible, including upscaling the chemical synthesis of polymers, and automating the conductor preparation procedures using techniques such as screen printing and laser cutting for better reproducibility and reliability.

[0127] Sensor application toolkit may be developed, to design a user-friendly application procedure for easy use of the technology, for improved deployability.

[0128] Application-targeted customization may be carried out to explore commercial value in specific applications and to tailor the material and sensor designs to better suit individual applications; and to engineer the signal acquisition system into a compact form factor and to provide complete hardware solutions (not only sensors) to specific applications.

[0129] Tn experiments carried out for plant electrophysiology monitoring and with reference to FIG. 13, dry conductive electrodes have poor conformability on hairy surface. Dry thermo-responsive polymer disclosed herein was able to maintain high signal quality under mechanical disturbance.

[0130] In experiments carried out for plant electrophysiology monitoring and with reference to FIG. 14A and FIG. 14B, long-term measurement of plant electrical signals over a month. Accurate measurement on plant circadian cycles when light / dark duration was changed.

[0131] Table 1 below provides a summary comparison of technologies.

[0132] TABLE 1 Comparison of technologies<

[0133] By “comprising” it is meant inciuding, but not limited to, whatever follows the word “comprising”. Thus, use of the term “comprising” indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0134] By “ consisting of’ is meant including, and limited to, whatever follows the phrase “consisting of’. Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0135] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including”, “containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0136] By “about” in relation to a given numerical value, such as for temperature and period of time, it is meant to include numerical values within 10% of the specified value.

[0137] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0138] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

CLAIMS1. A method of determining a state of a subject, the method comprisingapplying a liquid composition comprising a thermo-responsive polymer solvated therein to a surface of the subject,contacting a sensor with the liquid composition, anddrying the liquid composition to form a conformal thermo-responsive polymer layer, thereby affixing the sensor to the surface of the subject,wherein the conformal thermo-responsive polymer layer is operable as a capacitive coupling layer to allow capacitive acquisition of a signal from the subject by the sensor for determining the state of the subject.

2. The method according to claim 1, wherein the thermo-responsive polymer comprises or consists of one or both a polyethylene glycol / polypropylcnc glycol / polycaprolactonc urethane copolymer and a polyethylene glycol / polypropylene glycol / phenyl functionalized urethane copolymer.

3. The method according to claim 1 or 2, wherein the thermo-responsive polymer is one or more of a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and poly caprolactone (PCL) and connected with urethane bonds, and a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and 1 ,5-hexadiene-3,4-diol and connected with urethane bonds followed by thiol-ene click reaction with 2-phenylethanethiol.

4. The method according to any one of claims 1 to 3, wherein the thermo-responsive polymer comprises a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and polycaprolactone (PCL), connected with urethane bonds using hexamethylene diisocyanate (HMDI) as coupling reagent, and a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and l,5-hexadiene-3,4-diol and connected with urethane bonds using hexamethylene diisocyanate (HMDI) as coupling reagent followed by thiol-ene click reaction with 2-phenylethanethiol.5, The method according to any one of claims 1 to 4, wherein the sensor comprises an electrical conductor and contacting the sensor with the liquid composition comprises placing the electrical conductor in contact with the liquid composition.

6. The method according to claim 5, wherein the electrical conductor is one or more of silvcr / silvcr chloride, gold, carbon nanotubc, and a composite formed from carbon nanotubc and bacterial cellulose.7, The method according to any one of claims 1 to 6, wherein drying the liquid composition is carried out such that moisture content of the conformal thermo-responsive polymer layer remains essentially the same during the determining in an environment of intended usage.

8. The method according to any one of claims 1 to 7, wherein drying the liquid composition is carried out such that moisture content of the conformal thermo-responsive polymer is 5 wt% or less.

9. The method according to any one of claims 1 to 8, wherein the conformal thermo-responsive polymer layer is not ion conductive.

10. The method according to any one of claims 1 to 9, wherein the method further comprises obtaining one or more readings of electrical potential difference and / or electrical impedance as the signal over an extended time period of one month or more.

11. A kit for preparing a device for determining a state of a subject, the kit comprising a liquid composition comprising a thermo-responsive polymer solvated therein, wherein in use, the liquid composition forms a conformal thermo-responsive polymer layer on a surface of the subject upon disposing of the liquid composition on the surface of the subject with subsequent drying, wherein the conformal thermo-responsive polymer layer is operable as a capacitive coupling layer to allow capacitive acquisition of a signal from the subject for determining the state of the subject, anda sensor for affixing to the surface of the subject via the conformal thermo-responsive polymer layer.

12. The kit according to claim 11, wherein the thermo-responsive polymer comprises or consists of one or both a polyethylene glycol / polypropylene glycol / polycaprolactone urethane copolymer and a polyethylene glycol / polypropylene glycol / phenyl functionalized urethane copolymer.

13. The kit according to claim 11 or 12, wherein the thermo-responsive polymer is one or more of a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and poly caprolactone (PCL) and connected with urethane bonds, and a copolymersynthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and 1,5-hexadiene-3,4-diol and connected with urethane bonds followed by thiol-ene click reaction with 2-phenylethanethiol.14, The kit according to any one of claims 11 to 13, wherein the thermo-responsive polymer comprises a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and polycaprolactone (PCL), connected with urethane bonds using hexamethylene diisocyanate (HMDI) as coupling reagent and a copolymer synthesized with polyethylene glycol (PEG), polypropylene glycol (PPG) and l,5-hexadiene-3,4-diol and connected with urethane bonds using hexamethylene diisocyanate (HMDI) as coupling reagent followed by thiol-ene click reaction with 2-phenylethanethiol.15, The kit according to any one of claims 11 to 14, wherein the sensor comprises an electrical conductor for contacting the conformal thcrmo-rcsponsivc polymer layer.16, The kit according to claim 15, wherein the electrical conductor is one or more of silver / silver chloride, gold, carbon nanotube, and a composite formed from carbon nanotube and bacterial cellulose.17, Use of a method according to any one of claims 1 to 10 or a kit according to any one of claims 11 to 16 to obtain one or more readings of electrical potential difference and / or electrical impedance as a signal for determining a state of a subject over an extended time period of one month or more.

Images