Analyte determination method and sensing assembly

Abstract

A method is provided that includes providing a non-blood fluid sample having a concentration of an analyte, the analyte being individually detectable in blood. The method also includes determining an absolute or total amount magnitude of the analyte in the non-blood fluid, independent of any measurement of said concentration. Further provided is a sensing assembly for carrying out the method, and a method for determining a correlation between such magnitude of the absolute amount of the analyte in the non-blood fluid sample and the blood concentration of the analyte in blood.

Description

[Technical field]

[0001] The present invention relates to a method for the determination of an analyte, wherein the analyte is present in a non-blood bodily secretion sample, such as a sweat sample, and to a sensing assembly for carrying out said method using such a sample.

[0002] The invention further relates to a method for determining a correlation that can be used to determine the blood concentration of an analyte from a measure of the analyte determined using said non-blood fluid sample. [Background technology]

[0003] Non-invasive, semi-continuous, long-term monitoring of biomarkers indicative of disease / health and well-being is needed, for example, for dehydration, stress, sleep, child health monitoring and intraoperative monitoring.

[0004] Sweat, tears and saliva are all obtained non-invasively. Sweat is a particularly accessible biofluid and is a rich source of information about the subject's physiometabolism.

[0005] Some examples of clinically relevant components of sweat are Na+, Cl and / or K+ for monitoring dehydration, lactate as an early warning for inflammation (associated with sepsis), glucose for diabetes and neonates, and cortisol associated with sleep apnea and stress monitoring.

[0006] Continuous monitoring of high-risk patients, such as patients with severe chronic diseases, pre- or post-operative patients, and the elderly, using sweat biomarker monitoring devices can provide higher quality diagnostic information than periodic biomarker spot checks, as typically performed by repeatedly obtaining multiple blood samples. Such continuous monitoring can be performed in hospitals or other locations. Human sweat alone, or as a mixture with sebum lipids, is a readily available source for biomarker measurement in wearable on-skin devices. For example, cholesterol is an important biomarker associated with increased risk in the progression of cardiovascular disease. Inflammatory markers or cytokines, such as interleukins (e.g., TNF-a, IL-6), play an important role in immune responses and in detection or disease monitoring of joint damage in rheumatoid arthritis, psoriatic arthritis, and intestinal diseases.

[0007] Examples of biomarkers that can be detected in eccrine / apocrine sweat using appropriate capture species (antibodies, aptamers, molecularly imprinted polymers, etc.) are small molecules such as urea, creatinine, cholesterol, triglycerides, steroid hormones (cortisol), glucose, melatonin, peptides and proteins including cytokines such as IL-lalpha, IL-lbeta, IL-6, TNF alpha, IL-8 and TGF-beta IL-6, cysteine ​​proteases such as DNAse I, lysozyme, Zn-α2-glycoprotein, cysteine-rich secretory protein-3 and dermcidin, and large biomarkers such as hepatitis C virus.

[0008] As summarized by Mena-Bravo and de Castro in “Sweat: A sample with limited present applications and promising future in metabolomics”, J. Pharm. Biomed. Anal. 90, 139-147 (2014), there have been complaints about conventional sweat detection techniques regarding the difficulty of producing enough sweat for analysis, problems with sample evaporation, lack of suitable sampling devices, the need for trained staff, and problems with normalizing the amount sampled. Furthermore, sweat detection results have been found to be highly variable, and for various biomarkers, correlation between values ​​determined from blood and values ​​determined from sweat samples appears to be missing. In this regard, historical considerations in the field have included relatively crude sampling techniques, such as collecting large amounts of sweat in bags or fabrics. Deficiencies in such techniques may contribute to this apparent lack of correlation.

[0009] Efforts have been made to address these issues by bringing wearable sensors into near-instantaneous contact with sweat as it emerges from the skin. A recent example is the wearable patch shown in Gao et al., “Fully integrated wearable sensor arrays for multiplexed in situ perspiration analysis”, Nature 529, 509-514 (2016). The patch measures Na + , K + These include sensor arrays for measuring glucose, lactate and skin temperature. However, the focus of this work has been on the development and integration of the sensors themselves, and does not address the issue of collecting sweat samples, although this is clearly important. The latter is primarily due to the fact that there are several square centimetres (cm2) between the skin and the sensor. 2This has been done by placing an absorbent pad about the size of a 15 cm (0.2 in) on a surface of the body. The assumption is that if enough sweat is produced (hence the test is performed on exercising people), the pad will absorb the sweat for analysis, and the newly produced sweat will replenish the pad and "flush" away the old sweat. However, the time response characteristics of the sensor likely do not directly reflect the actual levels of the biomarker over time due to accumulation effects. Summary of the Invention [Problem to be solved by the invention]

[0010] Sample collection and presentation to published sensors may not be well controlled, making continuous reliable detection over long periods of time difficult, and such patches may not be designed to handle the small amounts of sweat normally produced, on the order of sub-nanoliters to nanoliters per minute per sweat gland. [Means for solving the problem]

[0011] The invention is defined by the independent claims. The dependent claims define advantageous embodiments.

[0012] According to one aspect, a method is provided comprising providing a non-blood fluid sample having a concentration of an analyte, the analyte being individually detectable in blood, and determining a magnitude of the absolute amount of the analyte in the non-blood fluid sample, independent of any measurement of the concentration.

[0013] The present disclosure is based on the insight that a more reliable correlation can be established between the analyte concentration in blood and the magnitude of the absolute amount of the analyte in a non-blood fluid sample compared to the scenario using the concentration of the analyte in a non-blood fluid sample.

[0014] In particular, since the absolute amount of analyte in the non-blood fluid sample is determined independent of any measurement of the concentration of the analyte in the non-blood fluid sample, it may not be necessary to measure the volume of the non-blood fluid sample and use this measured amount to derive the concentration of the analyte. What is important is to make the analyte determination measurement independent of variations in the volume of the non-blood fluid sample.

[0015] In some embodiments, the method further comprises calculating a magnitude of a blood concentration of the analyte in blood based on the determined magnitude of the absolute amount of the analyte in the non-blood fluid sample.

[0016] The blood concentration of an analyte tends to be considered the clinical standard and can be expressed, for example, in millimoles per liter (mM / l) or micromoles per liter (μM / l). The determined magnitude of the absolute amount of an analyte in a non-blood fluid sample can be used as a surrogate for the blood concentration of this analyte.

[0017] The calculating step can include using a correlation between the magnitude of the blood concentration and the magnitude of the absolute amount, which can be, for example, a look-up table, an analytical function, or the like.

[0018] In at least some embodiments, providing the non-blood fluid sample comprises obtaining the non-blood fluid sample from the subject's body over a predetermined period of time.

[0019] The given time period is selected according to the type of non-blood fluid sample and analyte, and is selected to ensure that the magnitude of the absolute amount of analyte determined in the non-blood fluid sample can be correlated with the blood concentration of the analyte.

[0020] The given time period may range, for example, from 5 minutes to 24 hours, for example, from 5 minutes to 90 minutes, for example, from 10 to 60 minutes, for example, about 15, about 30 or about 60 minutes.

[0021] For sweat being a non-blood secretion, the given time period in the above range serves to distribute the aforementioned time required for the analyte / biomarker to diffuse from the blood through the interstitial fluid, which serves to make the effect of this diffusion time negligible.

[0022] Any suitable total analytical technique may be used to determine the absolute magnitude of an analyte in a non-blood fluid sample, independent of the concentration of the analyte in the non-blood fluid sample.

[0023] The magnitude of the absolute amount of an analyte in a non-blood fluid sample may be expressed, for example, in grams or moles.

[0024] In some embodiments, the determining step comprises contacting the non-blood fluid sample with a capture species configured to selectively interact and / or react with the analyte.

[0025] Alternatively or additionally, the determining step comprises using a single molecule assay to detect a single molecule of the analyte.

[0026] The determining step using such a single molecule assay, for example a single molecule immunoassay, comprises counting the number of detected molecules of the analyte. In this way, a measurement of the absolute amount of the analyte can be made.

[0027] The capture species is selected according to the analyte of interest. Selective interaction and / or reaction between the capture species and small molecule analytes, such as lactate, urea, creatinine and cortisol, can be achieved, for example, using aptamers or molecularly imprinted polymers as the capture species.

[0028] In some embodiments, the analyte is Na + , Cl - , K + , NH4 + , H + , Ca 2+ Of particular interest are analytes, i.e. biomarkers, whose concentrations depend on the secretion / excretion rate of the non-blood fluid sample and / or whose dependence on the secretion / excretion rate is not currently known, i.e. they are NaCl, in the case where the non-blood fluid is sweat, + , Cl - , H + , lactate, cortisol, urea and Ca 2+ It is.

[0029] Additionally, there are analytes / biomarkers that are measured over a specific time frame to obtain a clinically meaningful measurement. Examples include, for example, urea, creatinine, NH4 + are renal markers such as

[0030] Some further examples of clinically relevant components of non-blood sweat secretions include NaCl for monitoring dehydration. + , Cl - and / or K + , lactate as an early warning for inflammation (associated with sepsis), glucose for diabetes and neonates, and cortisol associated with sleep apnea and stress monitoring.

[0031] In at least some embodiments, the non-blood fluid sample is a non-blood bodily fluid sample.

[0032] The non-blood fluid sample may be a sweat sample, or the non-blood fluid sample may be a saliva sample or a tear sample.

[0033] According to another aspect, a sensing assembly is provided having a sampling stage configured to receive a non-blood fluid sample having an analyte at a concentration, the analyte being individually detectable in blood, and an analysis stage configured to determine a magnitude of the absolute amount of the analyte in the non-blood fluid sample, independent of any measurement of the concentration.

[0034] In some embodiments, the sensing assembly further comprises a processing unit configured to calculate a magnitude of a blood concentration of the analyte in blood based on the determined absolute amount of the analyte in the non-blood fluid sample.

[0035] The processing unit is configured to calculate the blood concentration magnitude, for example using a correlation established between the blood concentration magnitude of the analyte and the absolute amount magnitude.

[0036] In some embodiments, the sampling stage is configured to receive the non-blood fluid sample for a given period of time, which, as described above with respect to the method, can be in a range such as, for example, 5 minutes to 24 hours, for example, 5 minutes to 90 minutes, for example, 10 minutes to 60 minutes, for example, about 15, about 30, or about 60 minutes.

[0037] Alternatively or additionally, the sampling stage may be positionable on or within the subject's body to receive a non-blood fluid sample.

[0038] The sampling stage may, for example, include a collection member for collecting a sample of the non-blood fluid over a period of time.

[0039] In a non-limiting example, the collection member comprises an absorbent material for receiving and absorbing the non-blood fluid sample from an area of ​​a subject's body over a period of time.

[0040] In another non-limiting example, the collection member has a membrane arranged to pass, e.g., allow, a non-blood fluid sample to pass therethrough, the membrane being activated to capture an analyte, the captured analyte being available for subsequent absolute quantity determination, e.g., at a later date, such activation of the membrane can be accomplished in any suitable manner, e.g., by a membrane having a molecularly imprinted polymer configured to selectively bind the analyte.

[0041] In some embodiments, the analytical stage comprises a capture species configured to selectively interact and / or react with the analyte, the capture species being placed in contact with the non-blood fluid sample.

[0042] Alternatively or additionally, the assay stage comprises a plurality of separate sensing regions, each sensing region arranged to detect a single molecule of the analyte.

[0043] In such embodiments, the analytical stage may also include a counter device configured, for example, to count the number of single molecules of analyte detected by each of the distinct sensing regions, thereby determining the magnitude of the absolute amount of the analyte in the non-blood fluid sample.

[0044] As described above with respect to the method, the analyte may be Na + , Cl - , K + , NH4 + , H + , Ca 2+ , lactate, ethanol, cortisol, glucose, urea or creatinine.

[0045] Alternatively or additionally, the non-blood fluid sample is a sweat sample, or the non-blood fluid sample may be saliva or tears.

[0046] According to yet another aspect, there is provided a wearable article comprising a sensing assembly according to any of the above-mentioned embodiments, which may further comprise, for example, a fixation element for fixing the sampling stage to a body of a subject, such fixation element may comprise, for example, a biocompatible adhesive and / or a strap suitable for attaching the sampling stage to a body of a subject.

[0047] According to a further aspect, Providing a plurality of blood samples; for each of the plurality of blood samples, providing a non-blood fluid sample obtained from the subject while obtaining one of the plurality of blood samples from the subject, each of the non-blood fluid samples having an analyte concentration; for each of said non-blood fluid samples, determining a magnitude of the absolute amount of said analyte in said respective non-blood fluid sample, independent of any measurement of said concentration in said respective non-blood fluid sample; providing a measure of the blood concentration of the analyte in each of the blood samples; and using the determined magnitude of the absolute amount of the analyte in each of the non-blood fluid samples and the determined magnitude of the blood concentration of the analyte in each of the blood samples to determine a correlation between the magnitude of the absolute amount of the analyte and the blood concentration of the analyte. A method is provided having the steps:

[0048] The embodiments described herein with respect to the method of determining correlation are applicable to the sensing assembly and method of determining the magnitude of the absolute amount of an analyte in a non-blood fluid sample, and similarly, the embodiments described herein with respect to the sensing assembly and method of determining the magnitude of the absolute amount of an analyte in a non-blood fluid sample are applicable to the method of determining said correlation. [Brief description of the drawings]

[0049] Embodiments of the invention will now be described in more detail, by way of non-limiting examples, with reference to the accompanying drawings, in which: [Figure 1] FIG. 1 provides a flow chart of a method according to one example. [Diagram 2] FIG. 2 illustrates a schematic of a sensing assembly according to an example. [Diagram 3] FIG. 3 shows a schematic of a sampling stage for collecting a non-blood fluid sample. [Figure 4] FIG. 4 shows a schematic diagram of a sampling stage according to another example. [Diagram 5] FIG. 5 shows a schematic of a core-shell particle in which the core of the particle carries a capture species. [Figure 6] FIG. 6 shows a schematic of an analytical stage having a carrier with a nanopore. [Figure 7] FIG. 7 provides a graph of the absolute amount of lactate in sweat samples versus the concentration of lactate in blood. [Figure 8] FIG. 8 provides a flow chart of a method for determining the correlation between the magnitude of the absolute amount of an analyte in a non-blood fluid sample and the blood concentration of the analyte in blood. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] While the detailed description and specific examples set forth exemplary embodiments of the devices, systems and methods, it should be understood that the description and examples are for illustrative purposes only and are not intended to limit the scope of the invention. These and other features, aspects and advantages of the devices, systems and methods of the present invention will become better understood from the following description, appended claims and accompanying drawings. It should be understood that the drawings are merely schematic and that the drawings are not drawn to scale. It should also be understood that the same reference numerals are used throughout the drawings to indicate the same or similar parts.

[0051] A method is provided which includes providing a non-blood fluid sample having a concentration of an analyte, the analyte being individually detectable in blood, the method also including determining an absolute or total amount of the analyte in the non-blood fluid sample independent of any measurement of the concentration, and further includes a sensing assembly for carrying out the method and a method for determining a correlation between such total amount of the analyte in the non-blood fluid sample and the blood concentration of the analyte in blood.

[0052] For analytes, i.e., biomarkers, that are present in both blood and non-blood fluids, it may be desirable to use the magnitude of the analyte content in such non-blood fluids as a surrogate for the blood concentration of the analyte.

[0053] Analyte blood concentrations tend to be considered the clinical standard. However, non-blood secretions, such as sweat, saliva, tears, etc., can be obtained less invasively than obtaining blood samples. Alternatively or additionally, sampling such non-blood secretions can facilitate long-term monitoring of subjects, especially outside of a medical environment. A significant challenge in utilizing the analyte content of such non-blood secretions as a surrogate for the analyte blood concentration is that the concentration of the analyte in the non-blood secretions does not reliably correlate with the blood concentration of said analyte.

[0054] For example, when considering sweat as a non-blood secretion, the concentrations of certain biomarkers have been shown to depend on sweat rate. Selected biomarkers present in sweat and their currently known dependence on sweat rate are shown in Table 1. [Table 1]

[0055] For biomarkers that can passively enter the sweat gland, dilution occurs at fairly high sweat rates due to the large amounts of sweat produced. Examples of such biomarkers are glucose, ethanol and cortisol. For example, for urea, a weak correlation with blood can be observed at fairly low sweat rates.

[0056] Because sweat rate changes more quickly than the biomarker and other molecules may find their way into sweat, the concentration of all molecules in sweat is essentially subject to the dilution described above. In addition, for some molecules, one or more additional factors may affect their concentration in sweat. The correlation between the concentration of a biomarker measured in sweat and the blood concentration of this biomarker can be explained by (i) the release of the biomarker (e.g., Na) from sweat as it leaves the sweat duct; + / Cl - (ii) production of the biomarker (e.g., lactate) by the glandular cells that produce the biomarker themselves; and (iii) the time it takes for the biomarker to diffuse from the blood through the interstitial fluid to the sweat gland. With regard to (iii), biomarkers may be reabsorbed from sweat, particularly due to filtration-induced movement of the biomarker from the blood towards the interstitial fluid and sweat gland (see below).

[0057] Mass transfer of fluid into and out of capillary beds, often referred to as bulk flow, occurs via two pressure-driven mechanisms: 1) the amount of fluid that moves from areas of higher pressure in the capillary bed to areas of lower pressure in the tissues via filtration; and 2) The movement of fluid from areas of higher pressure in tissues into areas of lower pressure in capillaries through reabsorption Two types of pressure, hydrostatic pressure and osmotic pressure, interact to drive each of these movements.

[0058] The main force that causes fluid transport between capillaries and tissues is hydrostatic pressure, which can be defined as the pressure of any fluid enclosed within a space. Blood hydrostatic pressure is the force exerted by blood trapped within a blood vessel or heart chamber. Even more specifically, the pressure exerted by blood on the walls of capillaries is called capillary hydrostatic pressure (CHP) and is the same as the blood pressure of the capillaries. CHP is the force that causes fluid to exit the capillaries and enter the tissues.

[0059] As fluid leaves the capillaries and moves into the tissues, there is a corresponding increase in hydrostatic pressure in the interstitial fluid. This opposing hydrostatic pressure is called interstitial fluid hydrostatic pressure (IFHP). Generally, the CHP resulting from the arterial pathway is much higher than the IFHP because lymphatics are continually absorbing excess amounts of fluid from the tissues. Thus, fluid generally tends to move out of the capillaries and into the interstitial fluid. This process is called filtration.

[0060] Capillary filtration and reabsorption occurs according to the equilibrium of hydrostatic and osmotic pressures, as described by the Starling hypothesis. Filtration force = k[(P c +π i )-(P i +π c )]

[0061] where k is the transmission coefficient, P c is the hydrostatic (blood) pressure in the capillaries, π i is the oncotic pressure in the interstitial fluid, P i is the hydrostatic pressure of the interstitial fluid, and π c is the oncotic pressure from plasma proteins in the capillaries. c and P i is "positive pressure" and π i and π c is a "negative pressure" (created by osmosis). The term filtration is used because red blood cells remain in the bloodstream, but some biomolecules migrate into the interstitial fluid.

[0062] It should be noted that certain biomarkers, e.g., low lipophilic (hydrophobic) analytes such as steroid hormones (cortisol, testosterone, etc.) and hydrophilic drugs (methylxanthines, levodopa, ethanol, etc.), show relatively strong correlations between sweat concentration and blood concentration. While such biomarkers are known to be partitioned transcellularly through lipophilic cell membranes, larger and / or more hydrophilic analytes, e.g., glucose, are speculated to enter sweat via paracellular routes, active channels or vesicular / exosomes, which confounds attempts to provide sweat concentration-blood concentration correlations. Furthermore, hydrophilic drugs, e.g., penicillin, ibuprofen and amoxicillin, are more likely to be diluted.

[0063] To alleviate the above problems, the present disclosure provides methods and apparatus for determining the magnitude of the absolute amount of an analyte in a non-blood fluid sample, such as a sweat sample, independent of any measurement of the concentration of the analyte in the non-blood fluid sample.

[0064] Compared to a scenario using the concentration of an analyte in a non-blood fluid sample, a more reliable correlation can be established between the blood concentration of the analyte and the thus determined magnitude of, for example, the absolute amount of the analyte in a non-blood fluid sample collected in a given time period.

[0065] In particular, the absolute amount of analyte in the non-blood fluid sample is determined independently of any measurement of the concentration of the analyte in the non-blood fluid sample, so there is no need to measure the volume of the non-blood fluid sample and use this volume to derive the concentration of the biomarker. What is important is to have a measurement that determines the analyte that is not dependent on changes in the volume of the non-blood fluid sample.

[0066] Using sweat samples as a representative example of non-blood fluid samples, it is noted that as exercise load increases, the blood concentration of a given analyte increases. However, at the same time, the concentration of the sweat analyte decreases (due to the dilution mentioned above due to the increased volume of sweat produced). However, as exercise load increases, the volume of sweat produced also increases, and this increase in volume may outweigh the decrease in sweat analyte concentration. As a result, the absolute amount, or total amount (i.e., amount multiplied by concentration), of the analyte in the sweat sample may also increase with exercise load, and thus may be correlated with the blood concentration, keeping in mind that blood volume remains constant.

[0067] It should also be noted that for such sweat samples, the amount of sweat is related to the number of active sweat glands, the sweat rate per gland, and the time period the sweat glands are active (i.e., amount = sweat rate x number of active glands x activation time).

[0068] In at least some embodiments, the magnitude of the absolute amount of analyte in a non-blood fluid sample taken from a particular reference body area, e.g., an area of ​​skin in the case of sweat sampling, during a given period of time is determined.

[0069] In some embodiments, as described in more detail herein below, such magnitude of the absolute amount of an analyte in a non-blood fluid sample is then compared or correlated to the blood concentration of that analyte.

[0070] 1 provides a flow chart of a method 100 according to one example. The method 100 includes providing 102 a non-blood fluid sample having an analyte concentration. The method 100 further includes determining 104 a magnitude of the absolute amount of analyte in the non-blood fluid sample, independent of any measurement of the concentration.

[0071] A non-blood fluid sample may be any suitable non-blood fluid if the analyte contained in the non-blood fluid sample is also detectable in the subject's blood, and thus the magnitude of the absolute amount of analyte in the non-blood fluid may be used as a surrogate for the blood concentration of said analyte in blood.

[0072] In some embodiments, the non-blood secretions are sweat, saliva, or tears, with sweat being particularly noted due to the ready availability of this biological fluid and the relevance of the analytes contained therein to the physiology and metabolism of the subject.

[0073] The analyte may be, for example, Na + , Cl - , K + , NH4 + , H + , Ca 2+ , lactate, ethanol, cortisol, glucose, urea and creatinine.

[0074] Importantly, it provides an analyte determination measurement that is not subject to variations in the volume of non-blood fluid samples; for example, analytes / biomarkers listed in Table 1 whose concentrations are secretion / excretion rate dependent and / or whose secretion / excretion rate dependence is not currently known are of particular interest; i.e., Na + , Cl - , H +, lactate, cortisol, urea and Ca 2+ It is.

[0075] Additionally, there are analytes / biomarkers that are measured over a specific time frame to obtain a clinically meaningful measurement. Examples are renal markers such as urea and creatinine.

[0076] In a non-limiting embodiment, creatinine, urea and / or NH4 in a sweat sample are + Determination of the absolute amount of can be replaced by determination of the same waste products in urine, for example monitored over a 24 hour period.

[0077] Some further examples of clinically relevant components of nonblood sweat secretions include NaCl for monitoring dehydration. + , Cl - and / or K + , lactate as an early warning for inflammation (associated with sepsis), glucose for diabetes and neonates, and cortisol associated with sleep apnea and stress monitoring.

[0078] 2 illustrates a schematic of an example sensing assembly 200. The sensing assembly 200 includes a sampling stage 202 configured to receive a non-blood fluid sample and an analysis stage 204 configured to determine a magnitude of an absolute amount of an analyte in the non-blood fluid sample independent of any measurement of the concentration of the analyte in the non-blood fluid sample.

[0079] In at least some embodiments, the providing step 102 comprises obtaining, or collecting, a non-blood fluid sample from a region of the subject's body, or from a reference region, over a given period of time.

[0080] The given time period is set according to the type of non-blood fluid sample and analyte, and is set such that the magnitude of the absolute amount of analyte determined in the non-blood fluid sample can be reliably correlated with the blood concentration of the analyte.

[0081] The given period of time may, for example, be in the range of 5 minutes to 24 hours, such as 5 minutes to 90 minutes, such as 10 to 60 minutes, for example about 15, about 30 or about 60 minutes.

[0082] In the case of sweat being a non-blood secretion, such a given time period may serve to extend the aforementioned time required for an analyte / biomarker to diffuse from the blood through the interstitial fluid, thus rendering the effect of this diffusion time negligible.

[0083] Referring to FIG. 2, a sampling stage 202 can be positioned, for example, on or within a subject's body to receive a non-blood fluid sample from an area 206 of the subject's body 208 over the given period of time.

[0084] In the instance where the non-blood fluid is sweat, the sampling stage 202 may be positioned on or proximate to the subject's skin to collect a sweat sample from the subject's skin.

[0085] More generally, the sensing assembly 200 may be included in a wearable article, which may further comprise a fixation element (not shown), for example, for fixing the sampling stage to the subject's body. Such a fixation element may comprise, for example, a suitable biocompatible adhesive and / or a strap for attaching the sampling stage to the subject's body.

[0086] In some embodiments, the providing step 102 includes collecting the non-blood fluid sample using a collection member 210. Such a collection member 210 can be included in the sampling stage 202 of the sensing assembly 200, for example.

[0087] In some embodiments, the collection member 210 comprises an absorbent material 212 for receiving and absorbing the non-blood fluid sample from the region 206 of the subject's body 208 over the given period of time. The magnitude of the absolute amount of analyte in the non-blood fluid sample so collected is then determined (104), as described in more detail herein below.

[0088] 2, the collection member 210 has an absorbent material 212 and a further absorbent material 214 for receiving and absorbing a further non-blood fluid sample for a further predetermined period of time. The collection member 210 may be configured such that the absorbent material 212 that collects the non-blood fluid sample is replaced with the further absorbent material 214 such that the further absorbent material 214 can receive and absorb the further non-blood fluid sample.

[0089] More generally, the sampling stage 202 may include a mechanism 216, e.g., having a rotatable drum, configured to move the absorbent material 212 away from the region 206 of the body 208 where the non-blood fluid sample is collected after the given period of time has elapsed.

[0090] In some embodiments, the mechanism 216 is configured to move the absorbent material 212 away from the area 206 of the body 208 where the non-blood fluid is collected and to an analysis stage 204 where the magnitude of the absolute amount of analyte in the non-blood fluid sample is determined (104).

[0091] In the non-limiting example shown in FIG. 2, the mechanism 216 can not only move the absorbent material 212 away from the region 206 of the body 208, but can also move additional absorbent material 214, as described above, into the region 206 of the body 208 vacated by the absorbent material 212.

[0092] In embodiments where the collection member 210 has an absorbent material 212 and a further absorbent material 214, the determination 104 of the absolute amount of analyte in the non-blood fluid sample collected in the absorbent material 212 is performed while the further absorbent material 214 is collecting the further non-blood fluid sample. An example of this is shown in FIG.

[0093] In a specific, non-limiting embodiment, the collection member 210 comprises an absorbent material and filter paper as a further absorbent material. In such an embodiment, a portion of the filter paper, corresponding to the absorbent material 212 described above, can collect a non-blood fluid sample, e.g., a sweat sample, during a given period of time. This portion of the filter paper is then transferred to the analysis stage 204, e.g., by mechanism 216 described above, while a further portion of the filter paper, corresponding to the further absorbent material 214 described above, collects a further non-blood fluid sample, e.g., a sweat sample.

[0094] The portions of filter paper may, for example, be separate or otherwise separate from one another, or, in other embodiments, the portions of filter paper may be included in a continuous roll from which unused portions of the filter paper are continuously exposed to the region 206 of the body 208 where the non-blood fluid is collected.

[0095] In some embodiments, the collection member 210 comprises a membrane through which, eg, is arranged to allow, a non-blood fluid sample, eg, sweat, to pass.

[0096] The membrane is activated to capture the analyte, which is then available, for example, at a later date, for subsequent determination of absolute quantity 104. Such membrane activation can be performed in any suitable manner, for example, by a membrane having a molecularly imprinted polymer configured to selectively bind the analyte.

[0097] For example, molecularly imprinted polymers are known for selectively binding lactate and cortisol, see, e.g., Alizadeh et al., Taianta 192 (2019) 103-111, and Parlak et al., Sci. Adv. 2018; 4 eaar2904.

[0098] Independent of any measurement of the concentration of the analyte in the non-blood fluid sample, the step of determining 104 the magnitude of the absolute amount of the analyte in the non-blood fluid sample may be performed in any suitable manner, for example, by contacting the non-blood fluid sample with a capture species configured to selectively interact and / or react with the analyte.

[0099] A typical determination 104 of a measurement of component A with capture species B can be expressed as A+B→AB. Such measurements can be made independent of the volume of the non-blood fluid sample by a) favoring the response to AB, for example, by increasing the amount of capture species and / or modifying the capture species to bind the analyte more strongly, or by moving AB away from A+B once formed. An alternative approach is b) to minimize or prevent the reversion of AB to A+B, for example, by using the size, conformation and / or charge changes that occur from A to AB.

[0100] More generally, the capture species is selected according to the analyte of interest. Selective interactions and / or reactions between the capture species and small molecule analytes such as, for example, lactate, urea, creatinine and cortisol can be achieved using, for example, aptamers or molecularly imprinted polymers as the capture species.

[0101] It is further noted that antibodies are known that can act as capture species to bind cortisol, see, e.g., Torrente-Rodriguez et al., Matter 2, 921-937, 2020.

[0102] In some non-limiting examples, the capture species may be or may comprise an enzyme. Such an enzyme can selectively bind to an analyte and, in some instances, convert the analyte in a chemical reaction. This binding, and in some instances, reaction, can be used in determining 104. For example, lactate can be selectively bound and converted by lactate dehydrogenase.

[0103] Where the analyte is an ion, the capture species may be in the form of an ion selective membrane, for example an ion selective membrane incorporated into an ion selective electrode.

[0104] In some non-limiting examples, the change in charge due to the binding of certain ions, such as sodium ions, changes the conformation and / or charge of the capture species, and this change is used to push the A+B→AB equilibrium towards AB.

[0105] In some embodiments, the analytical stage 204 of the sensing assembly 200 may include a sensing element that includes a capture species.

[0106] In embodiments that use a capture species in determining 104 , all or substantially all of a non-blood fluid sample, such as a sweat sample, may be provided to a sensing element of the analysis stage 204 .

[0107] The sensing element may have, for example, a capture surface onto which a capture species is provided, e.g., immobilized, and over which the non-blood fluid sample flows, or in some instances over which the non-blood fluid sample circulates until all or substantially all of the analyte has left the non-blood fluid sample and been captured by the capture species.

[0108] The amount of biomarker can be ascertained by any known method for measuring the interaction and / or reaction of the analyte with the capture surface, for example electrical, optical and / or magnetic measurement techniques.

[0109] It should be noted that in order to repeatedly perform decision 104, it may be necessary to provide a significant excess of capture species on the capture surface or for the capture surface to undergo a replenishment process, an example of which is described in WO2020 / 099570A1.

[0110] Alternatively, in the analysis stage 204, all or nearly all of the analyte is collected by a collection member 210, such as a membrane as described above, before determining 104 the absolute amount of analyte captured.

[0111] In such embodiments, the analysis stage 204 may use any suitable analytical principle for determining the absolute amount of the analyte. The determination 104 may be performed, for example, after the analyte is released from the capture species.

[0112] For example, chromatographic techniques can be used in which the elution of the analyte, or in some instances that still bound to the capture species, using a chromatographic column included in the analytical stage 204 is monitored by a suitable detector, and the height or integral of the detection signal corresponding to the analyte (or the analyte bound to the capture species) is used to derive the magnitude of the absolute amount of the analyte.

[0113] The chromatography column comprises one or more phases suitable for separating the analyte, or in some instances, the analyte that is still bound to the capture species, from other components present in the non-blood fluid sample.

[0114] For example, an internal standard can be used to assist in quantification of the analyte, eg, using the ratio of the area under the analyte signal to the area under the signal corresponding to the internal standard.

[0115] 3 shows an exemplary sampling stage 204 in the form of a wearable patch that is placed on the body 208, in this case the skin. The sampling stage 204 shown in FIG. 3 is thus used to collect sweat as a non-blood secretion.

[0116] In some embodiments, such as the non-limiting example shown in Figure 4, the analyte is captured in situ in a fluid system contained in the sampling stage 202, for example, near a region 206 of the body 208 where the non-blood fluid sample is to be collected, i.e., a sweat gland, using core-shell hydrogel particles 218. Such core-shell hydrogel particles 218 can have a capture species within the core of the particle surrounded by a hydrogel shell.

[0117] The core-shell hydrogel particles 218 can be contacted with a non-blood fluid sample and after a period of time, the particles can be transferred to the analytical stage 204 where a determination 104 is made.

[0118] For example, in the analysis stage 204, the analyte, or an analyte that has bound a capture species, such as an antigen, can be removed from the core-shell particles 218, for example by elution, and measured in a fixed volume.

[0119] Returning to Figure 4, the exemplary sampling stage 204 includes a collection member 210, for example in the form of a wearable patch as described above in connection with Figure 3, with core-shell particles 218 disposed within the collection member 210. The collection member 210 in this example may include or be in the form of an absorbent material 212 as described above.

[0120] A schematic diagram of a core-shell particle 218 is provided in Figure 5 showing a hydrogel shell 220 and a capture species 222 within the core of the core-shell particle 218. Additionally, the left pane of Figure 5 diagrammatically illustrates the core-shell particle 218 in contact with an analyte 224. In this particular example, the analyte 224 is associated with albumin 226, and the analyte 224 and albumin 226 are contained in a non-blood fluid sample.

[0121] The right pane of FIG. 5 shows that all of the analyte 224 has been bound by the capture species 220 within the core of the core-shell particle 218 , leaving the albumin 226 without the analyte 224 outside the core-shell particle 218 .

[0122] In some embodiments, an indicator approach is used in determining 104 the magnitude of the absolute amount of analyte in the non-blood fluid sample. Such an indicator approach requires that either all or substantially all of the non-blood fluid sample, e.g., sweat sample, be provided to a sensing element in the analytical stage 204, or that all or substantially all of the analyte be collected by a collection member 210, e.g., by a membrane as described above, before the absolute amount of analyte thus captured is determined 104 in the analytical stage 204.

[0123] In such an embodiment, determining 104 may include contacting the non-blood fluid sample, e.g., sweat sample, with an indicator material, e.g., an indicator liquid, configured to selectively interact and / or react with the analyte after a given period of time during which the non-blood fluid sample is collected. The absolute amount of the analyte may be determined 104 by any known method for measuring the overall reaction and / or interaction between the indicator material and the analyte, e.g., electrical, optical, or magnetic measurement techniques.

[0124] For example, reaction and / or interaction of the analyte with the indicator material causes the indicator material, e.g., an indicator liquid, to change color to enable, for example, the magnitude of the absolute amount of analyte in the non-blood fluid sample to be determined (104).

[0125] Another approach is to determine the absolute magnitude of the analyte in the non-blood fluid sample by measuring the total optical activity in a fluid mixture having an indicator material and the analyte in the non-blood fluid sample after a given period of time.

[0126] It should be noted that in order to make a repeat determination 104, it may be necessary either to provide a significant excess of indicator material compared to the specimen, or for the indicator material to be replenished, for example, by flushing the sampling volume and resuming sampling of non-blood fluid.

[0127] 6, the analytical stage 204 comprises a carrier 228 having a pore 230, in particular a nanopore 230. A detectable current 232 is provided through the pore 230. In such embodiments, the analytical stage 204 comprises a detector (not shown) configured to detect the current 232.

[0128] When the current 232 is blocked (eg, by molecules 234A, 234B, 234C), this is measured as a drop in the signal 236, as shown in FIG.

[0129] The nanopore 230 was initially developed for DNA sequencing and was later adapted to detect other molecules as well. Detection of specific analytes, particularly molecular analytes, can be done either i) by size exclusion, in which case the pore 230 allows only the molecule of interest to flow through and / or generates a specific signal, ii) by adding transport proteins that transport / recognize only the molecule of interest, or iii) by adding capture species to the pore 230 that induce an increase in residence time.

[0130] In each of these cases, the current is unidirectional, so the pore 230 essentially counts the total amount of analyte molecules that have passed through the pore 230. The current can be used to prevent any backflow of analyte, and / or proteins can be used to prevent such backflow.

[0131] More generally, determining 104 the magnitude of the absolute amount of analyte in a non-blood fluid sample can use counting methods, such as using a nanopore 230 configured to count each analyte molecule or ion as it is transported, or chemiluminescence, which involves counting the number of photons associated with each analyte molecule or ion. In such instances, the analyte molecules / ions may be removed after measurement.

[0132] Determining 104 the magnitude of the absolute amount of an analyte in a non-blood fluid sample can, in some embodiments, be performed using a single molecule assay, for example, a single molecule immunoassay.

[0133] In such single molecule assays (or alternatively so-called "digital assay" techniques), the analytical stage 204 includes a plurality of separate sensing areas, each separate sensing area, e.g., a detection well, configured to detect a single molecule of the analyte. In such embodiments, the analytical stage 204 includes a counter device configured to count the number of single molecules of the analyte detected by each of the separate sensing areas, thereby determining the magnitude of the absolute amount of analyte in the non-blood fluid sample.

[0134] In other words, the decision to use a single molecule assay 104 can include counting the number of detected molecules of the analyte. In this way, a measurement of the absolute amount of the analyte can be made.

[0135] It should be noted that such discretized sensing regions, when considered at a statistical / distributional level, can detect less than one analyte molecule of interest.

[0136] Examples of single molecule assays are described by Rissin et al., “Multiplexed single molecule immunoassays”, Lab Chip. 2013, 13(15):2902-11.

[0137] Regarding measurements, various known techniques can be integrated with the method according to the present disclosure.

[0138] For example, nanoparticles (eg, which may simply be referred to as "particles") can be used to deliver molecules into the detection wells described above, thereby providing a nanowell approach.

[0139] For example, nanoparticles are used to detect binding events: when the nanoparticle is in a region with zero or one molecule, the nanoparticle either exhibits Brownian motion interactions when no molecules are present, or exhibits a specific immobile state when one molecule is present.

[0140] This can be detected optically and enhanced by magnetic interactions.

[0141] An example of this is described by Ranzoni et al., “Frequency-Selective Rotation of Two-Particle Nanoactuators for Rapid and Sensitive Detection of Biomolecules”, Nano Lett. 2011, 11, 5, 2017-2022.

[0142] Another example of a single molecule assay is the proximity ligation assay (PLA), which produces a signal only when the molecule is present.

[0143] In another example, a conventional enzyme-linked immunosorbent-type assay can be used, provided that, for example, appropriate washing and / or separation steps are provided upstream of the analytical stage 204.

[0144] In at least some embodiments, the method 100 further comprises calculating 106 a magnitude of the blood concentration of the analyte in the blood based on the determined magnitude of the absolute amount of the analyte in the non-blood fluid sample.

[0145] In at least some embodiments, the calculation 106 includes using a correlation established between the magnitude of the blood concentration and the magnitude of the absolute amount.

[0146] Such a correlation is shown in graphical form in FIG. 7 for a specific, non-limiting example of absolute amount of lactate in sweat samples versus blood concentration of lactate.

[0147] This correlation may be, for example, in the form of a look-up table, an analytical function, or the like.

[0148] In some embodiments, the sensing assembly 200 includes a processing unit 238 configured to calculate a magnitude of the blood concentration of the analyte in the blood based on the determined magnitude of the absolute amount of the analyte in the non-blood fluid sample.

[0149] The processing unit 238 is configured to calculate the blood concentration magnitude using the above-mentioned correlation between the blood concentration magnitude and the absolute amount magnitude, which can be, for example, a look-up table, an analytical function, or similar form, as described above.

[0150] Further provided is a method 300 for providing the above-mentioned correlation, as shown in Figure 8. The method 300 comprises providing (302) a plurality of blood samples, and for each of the plurality of blood samples, providing (304) a non-blood fluid sample obtained from a subject at the time one of the plurality of blood samples is obtained from the subject.

[0151] The method 300 further comprises determining (306) for each of the non-blood fluid samples an absolute magnitude of the analyte in the respective non-blood fluid sample, independent of any measurement of the concentration of the analyte in the respective non-blood fluid sample, Determining 306 may be performed according to any of the examples described above with respect to step 104 of FIG. 1 and analysis stage 204 of FIG. 2.

[0152] The method 300 further includes providing 308, e.g., determining, a measure of the blood concentration of the analyte in each of the blood samples, which may utilize any known technique for determining the blood concentration of an analyte.

[0153] In step 310, a correlation between the magnitude of the absolute amount of the analyte in the non-blood fluid samples and the blood concentration of the analyte is determined using the determined magnitude of the absolute amount of the analyte in each of the non-blood fluid samples and the determined magnitude of the blood concentration of the analyte in each of the blood samples.

[0154] In this way, for example, a calibration curve of the type shown in FIG. 7 can be provided.

[0155] The disclosed devices, systems and methods have applications in non-invasive, semi-continuous, long-term monitoring of biomarkers indicative of health and well-being, such as dehydration, stress, sleep, child health monitoring and intra-operative monitoring. The devices, systems and methods are applicable to subject monitoring in general, but may also be particularly applicable to provide early warning for sudden deterioration of patients in general wards and intensive care units, or for investigating sleep disorders. It is also noted that currently, measurements are performed in a spot-check manner only when the patient is in the doctor's office, but the present disclosure may also be usefully employed in performing such spot-check measurements.

[0156] Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure and the appended claims. In the claims, the term "comprising" does not exclude other elements or steps, nor does it exclude a plurality if a plurality is not stated. Measures recited in mutually different dependent claims can be advantageously combined. Any reference signs in the claims should not be interpreted as limiting the scope thereof.

Claims

1. providing a non-blood fluid sample having a concentration of an analyte, said analyte being individually detectable in blood, said analyte being Na ＋ , Cl - , H ＋ , Ca ２＋ , lactate, cortisol, urea, or creatinine; and determining the magnitude of the absolute amount of said analyte in said non-blood fluid sample, independent of any measurement of said concentration. A method having the following.

2. The providing step includes obtaining the non-blood fluid sample from the subject's body over a predetermined time period, the method comprising:

10. The method of claim 1, further comprising calculating a magnitude of a blood concentration of the analyte in blood based on the determined magnitude of the absolute amount of the analyte in the non-blood fluid samples obtained from the subject's body over the predetermined time period.

3. The method of claim 2 , wherein the calculating step comprises using a correlation between the magnitude of the blood concentration and the magnitude of the absolute amount.

4. 10. The method of claim 1, wherein the providing step comprises obtaining the non-blood fluid sample from the subject's body over a predetermined period of time.

5. 10. The method of claim 1, wherein the determining step comprises contacting the non-blood fluid sample with a capture species configured to selectively interact and / or react with the analyte.

6. The method of claim 1 , wherein the determining step comprises using a single molecule assay to detect a single molecule of the analyte.

7. 10. The method of claim 1, wherein the analyte is lactate, cortisol, urea, or creatinine.

8. The method of claim 1 , wherein the non-blood fluid sample is a sweat sample.

9. a sampling stage configured to receive a non-blood fluid sample having a concentration of an analyte, the analyte being individually detectable in blood, the analyte being Na ＋ , Cl - , H ＋ , Ca ２＋ a sampling stage, which is lactate, cortisol, urea, or creatinine; and an analyzing stage configured to determine the magnitude of the absolute amount of said analyte in said non-blood fluid sample independent of any measurement of said concentration; a sensing assembly having

10. 10. The sensing assembly of claim 9, wherein the analysis stage is configured to obtain the non-blood fluid samples from the subject's body over a predetermined time period, the analysis stage having a processing unit configured to calculate a magnitude of a blood concentration of the analyte in blood based on the determined absolute amount of the analyte in the non-blood fluid samples obtained from the subject's body over the predetermined time period, and optionally the processing unit is configured to calculate the magnitude of the blood concentration of the analyte using a correlation between the magnitude of the blood concentration and the magnitude of the absolute amount.

11. the sampling stage is configured to receive the non-blood fluid sample over a given period of time; and / or the sampling stage being positionable on or within the subject's body to receive the non-blood fluid sample; The sensing assembly of claim 9 .

12. The detection assembly of claim 9, wherein the analytical stage comprises an excess of capture species configured to selectively interact and / or react with the analyte, the excess capture species configured to contact the non-blood fluid sample.

13. the analyte is lactate, cortisol, urea or creatinine; and / or The non-blood secretions are sweat. The sensing assembly of claim 9 .

14. A wearable article comprising the sensing assembly of claim 9.

15. providing a plurality of blood samples; For each of the plurality of blood samples, providing a non-blood fluid sample obtained from the subject while obtaining one of the plurality of blood samples from the subject, each of the non-blood fluid samples having an analyte at a concentration, the analyte being Na ＋ , Cl - , H ＋ , Ca ２＋ , lactate, cortisol, urea, and creatinine, and providing the non-blood secretory sample comprises obtaining the non-blood secretory sample from the subject's body over a predetermined period of time; for each of the non-blood fluid samples, determining a magnitude of the absolute amount of the analyte in each of the non-blood fluid samples, independent of any measurement of the concentration of the analyte in each of the non-blood fluid samples; providing a measure of the blood concentration of the analyte in the blood sample; and using the determined magnitude of the absolute amount of the analyte in each of the non-blood fluid samples and the determined magnitude of the blood concentration of the analyte in each of the blood samples to determine a correlation between the magnitude of the absolute amount of the analyte and the blood concentration of the analyte. A method having the following.

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