A sensing device for detecting an analyte in a liquid sample medium, a preparation apparatus for preparing an analyte containing liquid sample medium for use in such sensing device, a method for forming such sensing device and a method for detecting an analyte in a sample of air, in particular in a sample of respiratory air using such sensing device

Abstract

The present disclosure relates to a sensing device (100) for detecting an analyte in a liquid sample medium (50). Additionally, the disclosure also pertains to a preparation apparatus (200) for preparing an analyte containing liquid sample medium (50) for use in such sensing device (100). Furthermore, the present disclosure also pertains to a method for forming such sensing device (100) and a method for detecting an analyte in a sample of air, in particular in a sample of respiratory air using such preparation apparatus (200) and such sensing device (100).

Description

[0001] TITLE

[0002] A sensing device for detecting an analyte in a liquid sample medium, a preparation apparatus for preparing an analyte containing liquid sample medium for use in such sensing device, a method for forming such sensing device and a method for detecting an analyte in a sample of air, in particular in a sample of respiratory air using such sensing device.

[0003] TECHNICAL FIELD

[0004] The present disclosure relates to a sensing device for detecting an analyte in a liquid sample medium. Additionally, the disclosure also pertains to a preparation apparatus for preparing an analyte containing liquid sample medium for use in such sensing device. Furthermore, the present disclosure also pertains to a method for forming such sensing device and a method for detecting an analyte in a sample of air, in particular in a sample of respiratory air using such preparation apparatus and such sensing device.

[0005] BACKGROUND OF THE DISCLOSURE

[0006] In the history of diagnostics, breath analysis was one of the first method used until the breakthrough of biochemical testing technology. Today, breath analysis has made a comeback with the development of gas analysers and e-noses, demonstrating its power in its applicability for diagnosing a wide range of diseases. The technology is based on analysing exhaled volatile organic compounds (VOCs). Every time an individual breathes, thousands of volatile organic compounds (VOCs) and volatile fatty acids (VFAs), the breakdown products of lipids and proteins, are released into the breath stream. These novel biomarkers are by-products of metabolism of the human or animal body and can provide a snapshot of the subject’s health to identify food intolerances, guide asthma treatment decisions, early detection of gastroesophageal and lung cancer or breast cancer and diagnose of liver diseases or auto-immune disorders. Accordingly, the development of sensors towards the detection of these organic compounds has become an increasingly popular area of research over the last half a century. The presence of these compounds are key indicators of disease, environmental risks, and allow treatment if therapies are effective. Breath analysis has huge clinical potential, yet considerable work still needs to be performed to optimize sensing devices for collecting and analysing samples containing these organic compounds to be detected and moreover to accurately identify and analyse the VOC biomarkers to determine their predictive or prognostic value.

[0007] Accordingly, it is a goal of the present disclosure to provide an improved sensing device for detecting an analyte in a liquid sample medium, a method for forming such sensing device as well as a method for detecting an analyte in a sample of air, in particular in a sample of respiratory air.

[0008] SUMMARY OF THE DISCLOSURE

[0009] According to a first example of the disclosure, a sensing device for detecting an analyte in a fluid or liquid sample medium is proposed. The sensing device comprises a sensing chamber having an inlet for receiving the fluid or liquid sample medium containing the analyte as well as an outlet for discharging the fluid or liquid sample medium containing the analyte and a substrate having a substrate surface formulated to bind to the analyte when being flushed by the fluid or liquid sample medium containing the analyte. Additionally, a controller is provided which is structured to measure an impedance across the substrate, and structured to calculate an amount of the analyte bound to the substrate surface based at least in part on a change in impedance being measured.

[0010] This example provides a sensing device, which is fast, non-invasive, and requires no special expiration. This example according to the disclosure is easy to implement and is very feasible for use in children and elderly with limited breathing capacities. Additionally, due to its high sensitivity and selectivity, other diagnostic tests are unnecessary and thus the sensing device according to the disclosure will contribute to the reduction of doctor visits and referrals. Other possible applications are home monitoring of patients with chronic lung diseases and early detection of cancer. In general, the sensing device according to the disclosure, allows for an early onset of proper treatment and in addition can provide reassurance of 70% of parents that their child only suffers from a ‘viral wheeze’.

[0011] In a particular example, the substrate surface contains polycrystalline graphite or polycrystalline diamond, whereas in a more preferred example the polycrystalline graphite or the polycrystalline diamond is doped with boron. The use of polycrystalline graphite or polycrystalline diamond has proven to be advantageous to bind certain analytes or compounds which are contained in for example air and which provide a snapshot of the subject’s health to identify food intolerances, guide asthma treatment decisions, early detection of gastroesophageal and lung cancer and diagnose liver disease. This will contribute to better treatment and improved asthma control and will facilitate early detection of various types of cancer, with better treatment and improved prognosis as a consequence.

[0012] In a further example of the present disclosure, the substrate is selected from the group consisting of quartz, silicon dioxide or other ceramic materials. This results in an improved sensing platform with rigid construction.

[0013] In a further beneficial example, the controller comprises a pair of electrodes, with a first electrode positioned within the sensing chamber and the other, second electrode electrically connected with the substrate. Herewith, using the technique of electrochemical impedance spectroscopy (EIS) more analytical information can be acquired than with other analyzing techniques. EIS is most commonly used for the analysis of interfacial properties related to bio-recognition (e.g. antibody-antigen recognition, and substrate-enzyme interactions) at an electrodes surface, making it a powerful tool in the field of biomedical diagnosis.

[0014] Accordingly, with the controller implementing a pair of electrodes this technique is ideal for the analysis of all types of analytes binding to a functionalized surface, and offers critical details about the binding of the analyte (compound) to the surface of the substrate. In an example, the controller is capable of correctly determining the binding of e.g. acetaldehyde to the substrate surface.

[0015] In a preferred example, the first electrode is manufactured from a Group 11 material or an alloy thereof, in particular gold, whereas in another example the second electrode is formed as a base block manufactured from copper.

[0016] The controller of the sensing device according to the disclosure may furthermore comprises a heating element mounted to the substrate. In particular, the controller is structured to heat and maintain the sensing chamber at a predetermined temperature. Herewith the testing procedure can be executed under standardized temperature circumstances, ensuring a correct and reliable assessment of the analyte concentration in the liquid sample medium.

[0017] The present disclosure also pertains to a preparation apparatus for preparing an analyte containing liquid sample medium for use in the sensing device according to the disclosure. In an example, the preparation apparatus comprises a first holder for containing a certain amount of liquid sample medium; and a second holder for measuring a certain amount of air; an inlet tube having an inlet opening for receiving an air flow containing the analyte to be detected, and an outlet opening positioned inside the first holder and submerged below a surface of the certain amount of liquid sample medium; as well as a transfer tube for transferring air from the first holder into the second holder, the transfer tube having a transfer tube inlet opening positioned inside the first holder and above the surface of the certain amount of liquid sample medium, and a transfer tube outlet opening positioned inside the second holder and submerged below a surface of liquid contained in the second holder.

[0018] The platform technology according to the present disclosure is aimed at the detection of any analyte, for example analytes or compounds present in air. An example is the detection of acetaldehyde, being a natural product that is present in bread, coffee, and ripe fruit, but also a by-product of the partial oxidation of ethanol within the liver by alcohol dehydrogenase. Acetaldehyde is also commonly found in the air, fresh water sources, land and subsequently ground water. This prevalence of acetaldehyde has therefore led to numerous research lines studying the effects of acetaldehyde on the human body, with recent published work suggesting that the compound has strong links to asthma. It has been highlighted that higher blood concentrations of acetaldehyde induces bronchoconstriction, alongside increasing mite allergen-sensitized allergic inflammation in a murine model of asthma and is connected to the described symptoms.

[0019] Accordingly, the preparation apparatus according to the present disclosure aims at preparing a liquid sample medium for use in the sensing device according to the disclosure, in other words preparing a certain amount of liquid sample medium to absorb an amount of analyte usually contained in air, by flushing the liquid sample medium with air containing that analyte to be detected. In a preferred example of the preparation apparatus according to the present disclosure the second holder comprises an inverted measuring space and wherein the transfer tube outlet opening is positioned inside the inverted measuring space.

[0020] The disclosure also pertains to an assembly composed of the sensing device and the preparation apparatus as described in this patent application.

[0021] In addition, the present disclosure also relates to a method for forming a sensing device for detecting an analyte according to the disclosure, wherein the sensing device at least comprises a substrate having a substrate surface formulated to bind to the analyte. In particular, the method comprising at least the steps of: i) providing the substrate having a substrate surface containing polycrystalline graphite or polycrystalline diamond; ii) oxidizing the polycrystalline graphite or polycrystalline diamond containing substrate surface through immersion in a 3 / 1 mixture of sulphuric acid (H2SO4) and hydrogen peroxide (H2O2) ; iii) heating the mixture to 60 °C for 1 h; iv) rinsing the oxidized substrate with water and methanol; v) drying the oxidized substrate in an oven for 30 minutes at 65 °C; vi) immersing the oxidized surface in a solution of APTES in toluene (5% v / v); vii) heating the immersed oxidized surface to 75 °C for 2 h; viii) rinsing the oxidized surface with water and methanol and drying the oxidized surface in an oven for 30 minutes at 65 °C.

[0022] The method for detecting an analyte in a sample of air according to the disclosure is in particular suitable for detecting the analyte in a sample of respiratory air using the preparation apparatus according to the disclosure and subsequently the sensing device according to the disclosure.

[0023] The method may comprise the steps of guiding an air flow containing the analyte through a certain amount of liquid sample medium contained in the first holder of the preparation apparatus, allowing the analyte to be contained in the certain amount of liquid sample medium whilst measuring the amount of air flowing through the certain amount of liquid sample medium in the second holder of the preparation apparatus; subsequently passing the certain amount of liquid sample medium containing the analyte through the sensing chamber and over the substrate surface formulated to bind to the analyte; measuring an impedance across the substrate; and next calculating an amount of the analyte bound to the substrate surface based at least in part on a change in impedance being measured.

[0024] BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The disclosure will now be discussed with reference to the drawings, which show in:

[0026] Figure 1 an example of a sensing device according to the disclosure;

[0027] Figures 2A and 2B examples of a substrate for use in a sensing device according to the disclosure;

[0028] Figure 3 an example of a preparation apparatus according to the disclosure;

[0029] Figure 4A an electrochemical impedance analysis of both functionalized (dark colored line F) and non-functionalized (light colored line B) diamond substrates as a function of time with increasing concentrations of an analyte introduced;

[0030] Figure 4B the corresponding dose response graph belonging to Figure 4A showing the change in (A) effect size (%) of the functionalized layer vs the concentration of analyte (nM) introduced to the sensing device according to the disclosure;

[0031] Figure 5A an electrochemical impedance analysis (at 1.9 kHz) of the functionalized diamond substrate as a function of time with the introduced samples being exposed to acetaldehyde gas for increasing amounts of time;

[0032] Figure 5B the corresponding dose response graph showing the change in (A) effect size (%) vs the concentration of acetaldehyde introduced to the system as calculated from the volume, with data points representing the average of three measurements and the error bar being the standard deviation within those;

[0033] Figure 50 the calculated concentration of acetaldehyde present in solution based on the effect size vs the concentration of acetaldehyde gas bubbled through the system; Figures 6A an electrochemical impedance analysis (at 1.9 kHz) of the diamond substrate as a function of time with increasing concentrations (0.1 - 100 nM) of acetaldehyde in PBS that had been exposed to compressed air introduced;

[0034] Figure 6B the corresponding dose response graph showing the change in (A) effect size (%) of the functionalized layer vs the concentration of acetaldehyde (nM) introduced to the system, with the error bar representing the standard deviation within the measurements;

[0035] Figure 7A an electrochemical impedance analysis (at 1.9 kHz) of the diamond substrate as a function of time when exposed to breath samples and breath samples with increasing concentrations (0.1 - 100 nM) of acetaldehyde;

[0036] Figure 7B the corresponding response graph showing the change in (A) effect size (%) of the functionalized layer upon exposure to the breath and spiked breath introduced to the system, with the error bar representing the standard deviation within the measurements;

[0037] Figures 8A-8D different electrochemical impedance analyses (at 1.9 kHz) of the diamond substrate as a function of time when exposed to breath samples obtained from four persons, with P referring to the person and R referring to the repetition.

[0038] DETAILED DESCRIPTION OF THE DISCLOSURE

[0039] For a proper understanding of the disclosure, in the detailed description below corresponding elements or parts of the disclosure will be denoted with identical reference numerals in the drawings.

[0040] Every time an individual breathes, thousands of volatile organic compounds (VOCs) and volatile fatty acids (VFAs), the breakdown products of lipids and proteins, are released into the breath stream. These novel biomarkers are byproducts of metabolism of the human or animal body and can provide a snapshot of the subject’s health to identify food intolerances, guide asthma treatment decisions, early detection of gastroesophageal and lung cancer and diagnose liver disease.

[0041] The development of sensors towards the detection of these organic compounds has become an increasingly popular area of research over the last half a century. The presence of these compounds are key indicators of disease, environmental risks, and allow treatment if therapies are effective. Breath analysis has huge clinical potential, yet considerable work still needs to be performed to optimize sensing devices for collecting and analysing samples containing these organic compounds to be detected and moreover to accurately identify and analyse the VOC biomarkers to determine their predictive or prognostic value.

[0042] An example of an improved sensing device for detecting an analyte in a fluid or liquid sample medium is for example disclosed in Figure 1 , with further details in Figures 2A and 2B.

[0043] The sensing device for detecting an analyte in a fluid or liquid sample medium according to the disclosure is denoted with reference numeral 100. It comprises a microfluidic platform substrate formed as a sensing platform 101. The sensing platform 101 may comprise a spatial enclosure or sensing chamber 102. The sensing chamber 102 communicates with an inlet 101a for receiving a liquid sample medium 50 containing analytes x to be analyzed and also communicates with an outlet 101 b for discharging the liquid sample medium 50 containing analytes x as indicated by the bold arrows pointing to the right side of the Figure 1 , the direction of the arrows indicating the flow direction of the liquid sample medium 50 passing through the sensing platform 101 / sensing chamber 102.

[0044] The sensing chamber 102 of the sensing platform 101 is furthermore bound by a base block 106, which supports a first substrate side 103a of a substrate 103 inside the sensing chamber 102. The substrate 103 is provided with a functionalized substrate surface opposite to the first substrate side contacting the block 106, which functionalized substrate surface is indicated with reference numerals 104-104I-1042 (104n) in the various examples of Figures 1-2. The substrate 103 and its functionalized substrate surface 104-104I-1042 (104n) within the sensing chamber 102 will be - during operation of the sensing device 100 - flushed by the liquid sample medium 50 containing analytes x entering via the inlet 101a and leaving the sensing chamber 102 via the outlet 101 b. In particular, to ascertain an accurate measurement of the amount of analytes x, the sensing chamber 102 is filled with a certain amount of liquid sample medium 50 containing the analytes x, thereby submerging and wetting the substrate 103 and its functionalized substrate surface 104-104I-1042 (104n) for a particular period of time. According to the disclosure, the functionalized substrate surface 104- 104I-1042 (104n) of the substrate 103 is formulated to bind to analytes x being wetted by the liquid sample medium 50 containing the analytes x during that particular period of time. In a particular functional example, the functionalized substrate surface 104- 104I-1042 (104n) contains polycrystalline graphite or polycrystalline diamond as depicted in Figures 2A and 2B. More preferably, the polycrystalline graphite or the polycrystalline diamond is doped with boron. The use of polycrystalline graphite or polycrystalline diamond has proven to be advantageous to bind certain analytes or compounds which are contained in for example air and which provide a snapshot of the subject’s health to identify food intolerances, guide asthma treatment decisions, early detection of gastroesophageal and lung cancer and diagnose liver disease. This will contribute to better treatment and improved asthma control and will facilitate early detection of various types of cancer, with better treatment and improved prognosis as a consequence.

[0045] Likewise, in an example of the present disclosure, the substrate 103 is selected from the group consisting of quartz, silicon dioxide or other ceramic materials. This results in an improved sensing platform 101 with rigid construction.

[0046] Returning to Figure 1 , reference numeral 105 denotes a controller. The control 105 is capable to measure an impedance across the substrate 103. More in particular, the impedance measured across the substrate 103 is measured using a pair of electrodes 107a-107b, which are located at the opposite surface sides of the substrate 103, and which are electrically connected with the controller 105 in a suitable manner by means of control lines 107a-z and 107b-z. The first electrode 107a is preferable positioned within the sensing chamber 102 and is likewise - during operation - flushed by the liquid sample medium 50 containing analytes x. The other, second electrode 107b is electrically connected with the substrate 103. Herewith, using the technique of electrochemical impedance spectroscopy (EIS) more analytical information can be acquired that with other analyzing techniques. EIS is most commonly used for the analysis of interfacial properties related to bio-recognition (e.g. antibody-antigen recognition, and substrate-enzyme interactions) at an electrodes surface, making it a powerful tool in the field of biomedical diagnosis.

[0047] Accordingly, with the controller 105 implementing a pair of electrodes 107a-107b this technique is ideal for the analysis of all types of analytes x binding to a dedicated functionalized substrate surface 104n, which dedicated functionalized substrate 104nserves as one of the substrate sides of the substrate 103 across which surface sides the impedance is measured, and offers critical details about the binding of the analyte (compound) x to the surface 104nof the substrate 103. In an example, the controller is capable of correctly determining the binding of e.g. acetaldehyde to the substrate surface.

[0048] In a preferred example, the first electrode 107a reaching into the sensing chamber 102 is manufactured from a Group 11 material or an alloy thereof, in particular gold, whereas in another example the second electrode 107b is formed as the base block 106, which may be manufactured from copper.

[0049] The controller 105 is structured to calculate an amount of the analyte x bound to the substrate surface 104nbased at least in part on a change in impedance being measured between both electrodes 1-7a-107b.

[0050] This example provides a sensing device 100, which is fast, non-invasive, and requires no special expiration. This example according to the disclosure is easy to implement and is very feasible for use in children and elderly with limited breathing capacities. Additionally, due to its high sensitivity and selectivity other diagnostic tests are unnecessary and thus the sensing device according to the disclosure will contribute to the reduction of doctor visits and referrals. Other possible applications are home monitoring of patients with chronic lung diseases and early detection of cancer. In general, the sensing device according to the disclosure allows for an early onset of proper treatment, e.g. in children with asthma, and in addition can provide reassurance of 70% of parents that their child only suffers from a ‘viral wheeze’.

[0051] In a further beneficial example, the sensing device 100 may furthermore comprises a heating element 108 mounted to the substrate 106. In particular, the controller 105 is structured to heat and maintain the sensing chamber 102 at a predetermined temperature by controlling the heat element 108 via the control signal line 108-z. Herewith the testing procedure can be executed under standardized temperature circumstances, ensuring a correct and reliable assessment of the analyte x concentration in the liquid sample medium 50.

[0052] The present disclosure also pertains to a preparation apparatus for preparing an analyte containing liquid sample medium 50 for use in the sensing device 100 as depicted in Figures 1 and 2A-2B. In an example, the preparation apparatus 200 is shown in Figure 3. It comprises a first holder 201 which is suited containing a certain amount of liquid sample medium 50. Also a second holder 103 is provided for measuring a certain amount of air.

[0053] An inlet tube 202 having an inlet opening 202a for receiving an air flow 10 containing the analyte x to be detected with an initial concentration Cxi[-]. The inlet tube 202 has an outlet opening 102b, which positioned inside the first holder 201 and submerged below the surface 50z of the certain amount of liquid sample medium 50 contained therein.

[0054] Reference numeral 204 denotes a transfer tube for transferring air (see air bubbles 10 in the first holder 210) from the first holder 201 into the second holder 203. As shown in the Figure 3, the transfer tube 204 has a transfer tube inlet opening 204a positioned inside the first holder 201 and well above the surface 50z of the certain amount of fluid sample medium 50. A transfer tube outlet opening 204b is positioned inside the second holder 203 and submerged below a surface 60z of liquid 60 contained in the second holder 203.

[0055] The platform technology (sensing device 100 and the preparation apparatus 200) according to the present disclosure is aimed at the detection of any analyte x, for example analytes or compounds present in air 10. An example is the detection of acetaldehyde, being a natural product that is present in bread, coffee, and ripe fruit, but also a by-product of the partial oxidation of ethanol within the liver by alcohol dehydrogenase. Acetaldehyde is also commonly found in the air, fresh water sources, land and subsequently ground water. This prevalence of acetaldehyde has therefore led to numerous research lines studying the effects of acetaldehyde on the human body, with recent published work suggesting that the compound has strong links to asthma. It has been highlighted that higher blood concentrations of acetaldehyde induces bronchoconstriction, alongside increasing mite allergen- sensitized allergic inflammation in a murine model of asthma and is connected to the described symptoms.

[0056] Accordingly, the preparation apparatus 200 as shown in Figure 3 is structured to prepare a liquid sample medium 50 for us in the sensing device 100 according to the disclosure. The preparation apparatus 200 can be used to prepare a certain amount of liquid sample medium 50 contained in the first holder 201 to absorb an amount of analyte x contained in air 10, e.g. breathing air, by flushing the liquid sample medium 50 with air 10 containing that analyte x with an initial concentration of Cx1.

[0057] Preferably this is done, by allowing a user patient to blow breath 10 containing the analyte x from the lungs through the inlet tube inlet opening 202a of the inlet tube 202. The breath 10 contains the analyte x from the lungs with an initial concentration Cxileaves the inlet tube 202 via its inlet tube outlet opening 202b below the surface 50z of the liquid sample medium 50 causing bubbles 10’ in the liquid sample medium 50. The bubbles 10’ cause a fraction of the analyte x to dissolve in the liquid sample medium 50, the remaining fraction x will leave the first holder 201 with the air breath 10’ with a concentration CX2. Note that the concentration CX2 of the analyte x in the air 10’ passing through the transfer tube 204 is smaller that the concentration Cxiof the analyte x in the initial air sample 10 being blown by the user I patient through the inlet tube inlet 202.

[0058] As shown in the example of Figure 3, the second holder 203 of the preparation apparatus 200 comprises an inverted measuring space 205. The transfer tube outlet opening 204b is positioned inside the inverted measuring space 205 yet still submerged below the level 60z’ of the liquid 60 contained in the second holder 203. The inverted measuring space 205 is provided with a measuring scale 205z, which allows to measure the volume or amount of air 10’ being collected in the air chamber 205z above the level 60z’ of the liquid 60 in the inverted measuring space 205. This measured volume of air 10’ indicates the amount of air 10 being blown through the first holder 201 and the liquid sample medium 50.

[0059] It should be noted that the disclosure also pertains to an assembly composed of the sensing device 100 and the preparation apparatus 200 as described in this patent application as well as their combined use.

[0060] Their combined use can be defined as a method for forming a sensing device 100 for detecting an analyte x according to the disclosure, wherein the sensing device 100 at least comprises a substrate 103 having a substrate surface 104nformulated to bind to the analyte x. In particular, the method comprising at least the steps of: i) providing the substrate 103 having a substrate surface 104ncontaining polycrystalline graphite or polycrystalline diamond; ii) oxidizing the polycrystalline graphite or polycrystalline diamond containing substrate surface through immersion in a 3 / 1 mixture of sulphuric acid (H2SO4) and hydrogen peroxide (H2O2) ; iii) heating the mixture to 60 °C for 1 h; iv) rinsing the oxidized substrate with water and methanol; v) drying the oxidized substrate in an oven for 30 minutes at 65 °C; vi) immersing the oxidized surface in a solution of APTES in toluene (5% v / v); vii) heating the immersed oxidized surface to 75 °C for 2 h; viii) rinsing the oxidized surface with water and methanol and drying the oxidized surface in an oven for 30 minutes at 65 °C.

[0061] The method for detecting an analyte x in a sample of air 10 according to the disclosure is in particular suitable for detecting the analyte x in a sample of respiratory air 10 using the preparation apparatus 200 according to the disclosure and subsequently the sensing device 100 according to the disclosure.

[0062] The detection method may comprise the steps of guiding an air flow 10 containing the analyte x through a certain amount of liquid sample medium 50 contained in the first holder 201 of the preparation apparatus 200, allowing the analyte x to be contained or bound or dissolved from the air 10 into the certain amount of liquid sample medium 50 whilst measuring the amount of air 10’ flowing through the certain amount of liquid sample medium 50 in the second holder 203 of the preparation apparatus 200.

[0063] Subsequently the certain amount of liquid sample medium 50 containing the analyte x is passed through the sensing chamber 102 and over the substrate surface 104-104i-1042 (104n), the latter being formulated to bind to the analyte x. An impedance across the substrate 103 is measured and an amount of the analyte x bound to the substrate surface 104-104I-1042 (104n) is calculated based at least in part on a change in impedance being measured.

[0064] It should be noted that the development of all kinds of sensors towards the detection of simple organic molecules has become an increasingly popular area of research over the last half a century, with the presence of these molecules being key indicators of disease, environmental risks, and to access if therapies are effective. One such molecule of interest is acetaldehyde, being a natural product that is present in bread, coffee, and ripe fruit, but also a by-product of the partial oxidation of ethanol within the liver by alcohol dehydrogenase. Acetaldehyde is also commonly found in the air, having a low boiling point (20.2 °C), fresh water sources, land and subsequently ground water.

[0065] Below, and with the assistance of experimental date depicted in Figures 4A-4B, 5A-5C, 6A-6B, 7A-7B, and 8A-8D, the functionality of the sensing device 100 and the preparation apparatus 200 and in particular the effectiveness of the functionalized substrate 103-104 will be detailed and clarified.

[0066] As discussed, the prevalence of acetaldehyde has led to numerous research lines studying the effects of acetaldehyde on the human body, with recent published work strongly suggesting that the compound has strong links to asthma. The most common symptoms of asthma being chest tightness, sleep disturbance, nasal congestion, followed by breathlessness and wheezing. It has been highlighted that higher blood concentrations of acetaldehyde induces bronchoconstriction, alongside increasing mite allergen-sensitized allergic inflammation in a murine model of asthma and is connected to the described symptoms.

[0067] One area that shows potential in offering these advancements is the field of functionalized materials, where simple chemical modifications to a material substrate can yield high affinity surfaces for the specific binding and detection of a target analyte. To this end, numerous researchers have demonstrated this principle by introducing key chemical functionalities (proton donors / acceptors, lipophilic / hydrophilic groups) by the simple chemical pre-treatment of substrates, primarily with hydroxylating / oxidizing agents that facilitate a chemical anchor for the grafting of linker molecules containing more complex chemical architectures. This approach has been successful for the detection of gaseous small molecules such as acetone, hydrogen sulphide, and even acetaldehyde, though purely with the intent of environmental monitoring.

[0068] According to the disclosure, the functionalized substrate 103 with a substrate surface 104 formulated to bind to the analyte x, here acetaldehyde, are comprised of polycrystalline graphite or polycrystalline diamond. In a particular example, diamond substrate samples 103 were oxidized by immersing the diamond substrate sample 103 in a 3 / 1 mixture of sulphuric acid (H2SO4) and hydrogen peroxide (H2O2). This mixture was heated to 60 °C for 1 h. The resulting oxidized substrate 103 was then removed, rinsed with water and methanol, and dried in an oven for 30 minutes at 65 °C to afford the oxidized diamond substrate 103-104.

[0069] The oxidized surface 104I-1042 (104n) was then immersed into a solution of APTES in toluene (5% v / v) and heated to 75 °C for 2 h. The surface 104i- 1042 (104n) was then rinsed with water and methanol to remove any unbound material and salts, dried in an oven for 30 minutes at 65 °C, and collected to afford the resulting APTES-functionalized diamond substrate (D-APTES). The functionalized substrates were analyzed and characterized by means of contact angle, and XPS, see Figures 2A and 2B.

[0070] The functionalized diamond substrate 103-104i-1042 (104n) was placed inside a machined polycarbonate flow cell, as depicted in Figure 1. The polycarbonate flow cell I sensing platform 101 has a sensing chamber 102, as well as the inlet opening 101a and the outlet opening 101 b machined therein. Thus, the functionalized surface 104nis in direct contact with a liquid interface and the backside of the substrate 103 was in direct contact with a copper block 106, see also Figure 1.

[0071] Once sealed, the measurement cell (sensing device) 100 was heated to 37 °C using the controller 105 and the heating element 108 mounted to the copper block 106. This temperature maintained throughout the course of the analysis, ensuring a two minutes stabilization period prior to the measurement. Varying concentrations (between 0.1 - 100 nM) of acetaldehyde in PBS (x1) were then infused via the inlet opening 101a into the flow cell 101 , preferably at a rate of 21 mL I min for 10 seconds and at defined time intervals (every 200 seconds).

[0072] The impedimetric response (between 10 Hz and 0.5 MHz) across the functionalized layer 103-104nwas monitored by means of a gold electrode 107a placed in the sensing chamber 102 of the flow cell 101 and the counter electrode 107b being the copper block 106. For comparison the same acetaldehyde solutions were exposed to compressed air and the same analysis performed, thereby evaluating the effects of competitive gasses on the functionalized layer 103-104nof the measuring device 100.

[0073] The response of the functionalized diamond substrate 103-104nwas analyzed, however the preparation of the acetaldehyde solutions introduced to the measurement cell differed. To this end, acetaldehyde gas was bubbled through (Flow rate = 50 mL min-1 , Pressure = 2.5 bar) PBS (10 mL) for 10, 20, 30, 40, 50 and 60 seconds facilitating the gas to be dissolved into the aqueous medium 50 using the preparation apparatus 200 of Figure 3. After subjugation to the gas 10, the solutions were sealed in airtight vials and wrapped with film ensuring the short term longevity of the samples. Prior to analysis the samples were transferred to 10 mL syringes enabling the direct infusion of the samples via the inlet opening 101a into the measurement device 100.

[0074] Both functionalized diamond substrates 1042 and non-functionalized diamond substrates 104i were analysed by means of both contact angle and XPS measurements, enabling a comparison of the two materials. It is clear from the contact angle measurement that the functionalized substrate 1042 has greater hydrophobicity with the contact angle increasing from 49.62° for the non-functionalized substrate 104i to 98.46° for the functionalize substrate 1042 upon the introduction of a drop of water, as depicted in Figures 2A and 2B. This increase is logical with the increased hydrophobicity stemming from the introduction of alkyl chains, and subsequent masking of the polar hydroxyl groups that would natively be found at the surface. The APTES introduced also contains a polar amine functionality, though this is inconsequential when considering the coupling of the sillyl group to the surface requires three hydroxyl groups to install just one alkyl chain containing an amine. Overall leading to a decrease in the amount of available polar functionalities to interact directly with the water and therefore increasing the lipophilic nature of the substrate.

[0075] To further solidify the functionalization of the diamond, XPS studies were performed to understand the elemental composition of the samples. Both blank and functionalized samples were analysed for a direct comparison of the functionalization process. Table 1 contains the full elemental breakdown of the samples, highlighting that there are some major differences between the two samples. The primary difference lies in the percentage of carbon, nitrogen, oxygen, and silicon that the samples exhibit. The non-functionalized material has a higher carbon content stemming from the fact that the surface is mainly unreacted diamond (carbon), whereas the functionalized material shows a lower overall carbon content and an increase silicon count. This observation is in line with what is to be expected, as the surface is being modified with APTES that contains a greater silicon content. Both nitrogen and oxygen percentages in the functionalized substrates are also seen to rise, once again being indicative of the chemical process that the diamond substrate has been subjected to. The APTES is the source of nitrogen, but the prior oxidation of the surface will yield the overall increase in oxygen content of the sample.

[0076] Table 1. Elemental Composition [in atom-%; normalized to 100%] of diamond samples. MP1 and MP2 refer to two measurement points (1 and 2) on each sample.

[0077] These analyses show that the functionalization of the diamond substrates 103-104naccording to the disclosure are effective, with a clear increase in both the contact angle and the percentage silicon, nitrogen and oxygen of the samples that falls in line with what should be expected if the APTES was introduced to the surface of the material.

[0078] The analysis of (liquid-liquid) acetaldehyde dose response of the nonfunctionalised diamond substrates 104i (depicted in Figure 2A) and functionalised diamond substrate 1042 (depicted in Figure 2B) to the presence of acetaldehyde (= analyte x) was determined by impedimetric analysis according to the disclosure. To this end, increasing concentrations (0.1 - 100 nM) of acetaldehyde in PBS were introduced (= flushed) through the sensing chamber 102 and across the substrates 104i and 1042 and their electrochemical impedance was monitored between 10 Hz and 0.5 MHz, with the liquid-surface interaction being determined by the analysis of the associated BODE plots where the phase of the analysis fell between -40° and -50°.

[0079] The impedance as measured by the controller 105 was plotted in Figure 4A as a function of time before being converted into an effect size for a direct comparison between the non-functionalised substrate material 104i (line B) and functionalised substrate material 1042 (line F). In this graph, several measured impedance peaks are shown to arise over time, wherein each measured impedance peak is caused due to turbulent behaviour of the liquid sample medium 50 during flushing of the sensing chamber 102. In particular, the sensing chamber 102 is filled with liquid sample medium 50 containing the analytes x and consequently the substrate 104 is wetted by the liquid sample medium 50 during a certain period of time. During this period of time, analytes x can bind to the substrate surface 104I-1042, resulting in a change in the impedance measured over the substrate 103, which results in the measured impedance peaks in Figure 4A. Both substrates 104I-1042 according to the configuration of Figures 2A and 2B show a response towards the presence of the acetaldehyde, however the response of the functionalised substrate 1042 (Figure 2B) is greater than that of the non-functionalised substrate 104i (Figure 2A).

[0080] Throughout the concentration regime there is a clear difference between the raw diamond 104i and functionalised diamond 1042, indicating that the presence of the extra functionality (APTES) has added benefit towards the specific binding the acetaldehyde to the surface of the substrate 103-1042. This can be rationalised as the presence of the introduced amine functionality can form hydrogen bonds with hydrogen present in the aldehyde, and the aldehyde carbonyl can form hydrogen bonds with the hydrogen present in the amine at the functionalised surface.

[0081] In addition, the presence of the APTES alkyl chain may also help attract and bind the acetaldehyde, as there is increasing van der Waals interactions between the two species in comparison to the aldehyde and the raw diamond 104i. The observed change proceeded to be plotted as a function of concentration, enabling the generation of the corresponding dose response, see Figure 4B. The plotted data was fitted linearly (y = ax + b) (straight line), yielding a R2= 0.97918. This indicates a strong correlation between the acetaldehyde concentration and the observed change in effect size. This data therefore demonstrates that the sensing device 100 according to the disclosure shows potential towards the sensing of acetaldehyde as it is clearly capable of showing a quantifiable response in the presence of increasing analyte concentrations. The response seems to be linear in nature, as gives the developed functional substrate that has potential at operating within physiological concentration. As the sensing device 100 according to the disclosure is aimed at detecting the presence of acetaldehyde in breath (gas), the sensing device 100 according to the disclosure must also be scrutinized in a manner where gaseous acetaldehyde is dissolved into a liquid that can then be analysed. This therefore facilitates an understanding how the sensing device 100 will perform under such conditions and enables a comparison between the liquid-liquid preparation of acetaldehyde solutions and the gas-liquid preparation.

[0082] The analysis of the (gas-liquid) acetaldehyde dose response was therefore performed by bubbling acetaldehyde gas through PBS for defined amounts of time (0 - 60 seconds), before converting the volume passed through the solution into a concentration. As before, the raw electrochemical impedimetric data as obtained by the controller 105 was converted into an effect size and plotted as a function of time.

[0083] The data as shown in Figure 5A reveals a steady increase in the effect size as acetaldehyde is bubbled for greater time through the PBS solution. Thus, indicating that as the more gas is bubbled through the solution the amount of dissolved acetaldehyde is seen to be increasing. This theory is bolstered by plotting the effect size vs the concentration of acetaldehyde bubbled through the solution.

[0084] A clear linear relationship between the two variables can be seen, with the fit data (straight line, y = ax + b, R2 = 0.99858) of Figure 5B having a near perfect correlation coefficient suggesting a strong relationship between the acetaldehyde concentration introduced and the effect size observed. At the lower (0.2 M) and upper (1.4 M) concentration, the standard deviation (STDEV) in the measured response is greater indicating that the system operates more optimally between these points as the STDEV is greatly reduced.

[0085] As the relationship of the fitting curve of Figure 5B is linear, the full dynamic range of the sensing device 100 has not fully been reached as there no observable saturation affect, however as the concentration of acetaldehyde in breath is much lower than the concentration introduced this fact is inconsequential. This instead highlights how the sensing device 100 can be used to detect acetaldehyde in liquid samples that have had (acetaldehyde containing) gas bubbled through them (gas to liquid). It is thus demonstrated that the sensing device 100 according to the disclosure is capable of operating with liquid samples 50 that have had the target analyte x introduced in such a fashion. To further understand the amount of acetaldehyde transferred / dissolved into the medium 50 through bubbling using the preparation apparatus 200 according to the disclosure, the observed effect size was converted into an acetaldehyde concentration using the dose response previously generated in the liquid-liquid experiments, see Figure 5B. The amount of acetaldehyde dissolved in solution was then plotted against the concentration of acetaldehyde bubbled through the solution (see Figure 5C), enabling the relationship between the two variables to be studied.

[0086] As a greater concentration of acetaldehyde is bubbled through the liquid sample medium solution 50, the amount of acetaldehyde dissolved into the liquid sample medium solution 50 is grows as well (as expected). The relationship between these two variables is non-linear, with greater concentrations of bubbled acetaldehyde leading to a greater concentration of acetaldehyde dissolved into the liquid sample medium solution 50. It is assumed that there is a point where the amount of analyte x dissolved can no longer increase as the liquid sample medium solution 50 becomes saturated.

[0087] When comparing the amount of moles introduced to the amount of moles dissolved, there is a factor 1000 difference between the concentrations Cxiand CX2 of the analyte concentration in the air 10 and the air 10’ (see Figure 3). Thus, to get a sufficient amount of acetaldehyde (analyte x) into the liquid sample medium solution 50 to be detected by the sensing device 100, there has to be an elongated exposure time, or a higher concentration present in the gas form (air or breath 10).

[0088] In Figures 6A and 6B an analysis of acetaldehyde in the presence of compressed air is shown, as further considerations were made towards the complex nature of breath, with the constituent components being a reflection of the gasses that can be found in the air. It was therefore a logical set to expose PBS solutions containing acetaldehyde to compressed air, acting as a close representation of the chemical composition of breath and enabling the study of the effect of the gasses on the sensing device’s performance. To this end, the same concentration regime of acetaldehyde was used as per previous in the liquid-liquid analysis, facilitating the direct comparison of the obtained results and enabling the effects of the gasses to clearly observable.

[0089] Figure 6A shows an electrochemical impedance analysis (at 1.9 kHz) of the diamond substrate as a function of time with increasing concentrations (0.1 - 100 nM) of acetaldehyde in PBS that had been exposed to compressed air introduced, whereas Figure 6B depicts the corresponding dose response graph showing the change in (A) effect size (%) of the functionalized layer vs the concentration of acetaldehyde (nM) introduced to the system, with the error bar representing the standard deviation within the measurements.

[0090] As with the other experiments the sensing device 100 was initially stabilised in PBS, providing a standardised starting point (Figure 6A). Upon the addition of the PBS that had been exposed to compressed air (CA) the effect size was seen to rapidly drop, indicating that the presence of bubbled gasses has a major effect on the system. In contrast to acetaldehyde, the CA has an opposing effect decreasing the observed effect size and supressing the signal. By analysing the raw data, it is apparent that this change is conductive as the impedance is seen to be decrease, whereas the acetaldehyde affords a resistive change.

[0091] This fact therefore has a knock-on effect throughout the rest of the measurement, with the addition of acetaldehyde having a decreased resistive impact on the sensing device that can clearly been seen through the diminished effect sizes. For comparison, the addition of 0.1 nM acetaldehyde in this experiment generated a response of roughly 1%, whereas in the absence of the CA the response was double at 2%. This trend continues with each addition, with the final addition of 100 nM yielding a response of 3% in comparison to 8%.

[0092] When extrapolating these values and plotting the response against the concentration in Figure 6B, the data proves to be less linear with a greater overall standard deviation, and a linear fit yielding an R2 of 0.93772. It is therefore clear that the presence of other gasses in solution have a dramatic effect on the performance of the sensing device, and this has to be taken into consideration when performing more complex analysis of real samples.

[0093] To verify if the sensing device 100 according to the disclosure is capable of working with solutions that have been exposed to breath (reference numeral 10 in Figure 3, a “breath bubbling” setup was devised using the preparation apparatus 200 of Figure 3. Breath 10 was bubbled through a defined volume of PBS (500 mL) (= liquid sample medium 50) that was contained inside a bubble trap formed by the first holder 201 of Figure 3, and the overall volume of breath exhaled (500 mL) was monitored by the inverted measuring space 205 mounted in the second holder 203, with the liquid 60 being water (H2O).

[0094] Thus, the volume of breath 10 passed through the preparation apparatus 200 was determined by the volume of water 60 displaced from the inverted the inverted (cylinder) measuring space 205. Samples of the “breath PBS” were then analysed in same manner as previously described, introducing the raw “breath PBS” and acetaldehyde spiked (0.1 , 1 , 10, 100 nM) across the functionalized diamond layer 103- 104nof the measuring device 100 and analysing the change in electrochemical impedance.

[0095] The electrochemical impedance analysis (at 1.9 kHz) of the diamond substrate 103-104 according to the disclosure as a function of time when exposed to breath samples and breath samples with increasing concentrations (0.1 - 100 nM) of acetaldehyde is depicted in Figure 7A, whereas Figure 7B depicts the corresponding response graph showing the change in (A) effect size (%) of the functionalized layer upon exposure to the breath and spiked breath introduced to the system, with the error bar representing the standard deviation within the measurements.

[0096] The introduction of both the “breath PBS” and the spiked samples stimulate a response by the sensing device 100 as previously seen with both the liquidliquid and gas-liquid measurements. The collected results were compared to that of the dose response conducted after exposure to CA, with the same initial PBS step for a standardized comparison. The initial introduction of the “breath PBS” stimulated a 0.48% effect size that correlates to be a concentration of 0.077 nM when calculated from the CA dose response.

[0097] This value is a factor 2 lower than the physiological value found in breath (0.2 - 0.6 nM), indicating that the sensing device is under performing or something further is suppressing the observed signal. As the composition of CA and breath are very similar, it would be a logical jump to assume that the presence of organic molecules within the breath may also be causing a suppressed signal. Further evidence of this assumption is supported by the addition of the spiked samples, with 0.1 nM and 100 nM stimulating similar changes in effect size (A) as seen in the CA dose response.

[0098] When comparing the overall effect size (2.5 %) with the prior measurement, it can be seen that the signal is marginally suppressed (0.5%). According to these results, the sensing device 100 according to the disclosure is able to accurately identify and analyse the presence of acetaldehyde (captured from breath). Accordingly, these results can assist to identify food intolerances, guide asthma treatment decisions, early detection of gastroesophageal and lung cancer and diagnose liver disease.

[0099] Figures 8A-8D reveal a further assessment of the performance of the sensing device according to the disclosure. They show different electrochemical impedance analyses (at 1 .9 kHz) of the diamond substrate as a function of time when exposed to breath samples obtained from four persons, with P referring to the person and R referring to the repetition.

[0100] In particular, four healthy volunteer’s breath was collected and exposed to the sensing device 100 in triplicate (left, middle and right graphs of each of the Figures 8A-8D). As can be seen across the data set, the back calculated values of acetaldehyde in the samples were found to be between 0.0318 - 0.0828 nM. These values are in the same range as the prior experiment demonstrating the consistency of the sensing devices, but also reiterating the suppressive effects of other components in the breath.

[0101] Of the samples analysed two thirds performed as expected, with only four (the boxed graphs) of the samples producing results outside the expected ranges. Samples P2R1 , P3R1 and P4R1 show signal drift resulting in a negative delta, whereas P2R3 was observed to generate a signal with high noise that most likely stemmed from the presence of air bubbles within the analysis equipment. The other measurements all demonstrate the same trend, generating delta values that correspond with acetaldehyde concentration in line with the previous measurement. The fact that these values are all a two-fold decrease compared to literature values adds certainty to the results, but also opens up the possibility of adding a correction factor to the data set to bring it in line with physiological concentrations. LIST OF REFERENCE NUMERALS USED

[0102] 50 fluid or liquid sample medium

[0103] 50z surface of fluid or liquid sample medium in first holder 201

[0104] 60 liquid contained in second holder 203

[0105] 60z surface of liquid 60 contained in second holder 203

[0106] 60z’ surface of liquid 60 contained in air chamber 205z x analyte

[0107] Cxi concentration 1 of analyte x

[0108] CX2 concentration 2 of analyte x

[0109] 100 sensing device according to the disclosure

[0110] 101 sensing platform

[0111] 101a inlet opening

[0112] 101 b outlet opening

[0113] 102 sensing chamber

[0114] 103 substrate

[0115] 103a first surface side

[0116] 104-104nexamples of substrate surface formulated to bind to the analyte x

[0117] 105 controller

[0118] 106 base block

[0119] 107a / 107b first electrode / second electrode

[0120] 107a-z / 107b-z signal / control line of first electrode I second electrode

[0121] 108 heating element

[0122] 108z signal / control line of heating element

[0123] 200 preparation apparatus

[0124] 201 first holder

[0125] 202 inlet tube

[0126] 202a inlet tube inlet opening

[0127] 202b inlet tube outlet opening

[0128] 203 second holder

[0129] 204 transfer tube

[0130] 204a transfer tube inlet opening

[0131] 204b transfer tube outlet opening 205 inverted measuring space

[0132] 205x measuring scale

[0133] 205z air chamber of inverted measuring space

Claims

CLAIMS1. A sensing device for detecting an analyte in a liquid sample medium, comprising: a sensing chamber having an inlet for receiving the liquid sample medium containing the analyte as well as an outlet for discharging the liquid sample medium containing the analyte and a substrate having a substrate surface formulated to bind to the analyte when being flushed by the liquid sample medium containing the analyte; as well as a controller structured to measure an impedance across the substrate, and structured to calculate an amount of the analyte bound to the substrate surface based at least in part on a change in impedance being measured.

2. The sensing device according to claim 1 , wherein the substrate surface contains polycrystalline graphite or polycrystalline diamond.

3. The sensing device according to claim 2, wherein the polycrystalline graphite or the polycrystalline diamond is doped with boron.

4. The sensing device according to any one of more of the preceding claims, wherein the substrate is selected from the group consisting of quartz, silicon dioxide or other ceramic materials.

5. The sensing device according to any one of more of the preceding claims, wherein the controller comprises a pair of electrodes, with a first electrode positioned within the sensing chamber and the other, second electrode electrically connected with the substrate.

6. The sensing device according to claim 5, wherein the first electrode is manufactured from a Group 11 material or an alloy thereof, in particular gold.

7. The sensing device according to claim 5 or 6, wherein the second electrode is formed as a base block manufactured from copper.

8. The sensing device according to any one of more of the preceding claims, wherein the controller furthermore comprises a heating element mounted to the substrate, wherein the control is structured to heat and maintain the sensing chamber at a predetermined temperature.

9. A preparation apparatus for preparing an analyte containing liquid sample medium for use in the sensing device according to any one of more of the preceding claims, wherein the preparation apparatus comprises:a first holder for containing a certain amount of liquid sample medium; and a second holder for measuring a certain amount of air; an inlet tube having an inlet opening for receiving an air flow containing the analyte to be detected, and an outlet opening positioned inside the first holder and submerged below a surface of the certain amount of liquid sample medium; as well as a transfer tube for transferring air from the first holder into the second holder, the transfer tube having a transfer tube inlet opening positioned inside the first holder and above the surface of the certain amount of liquid sample medium, and a transfer tube outlet opening positioned inside the second holder and submerged below a surface of liquid contained in the second holder.

10. The preparation apparatus according to claim 9, wherein the second holder comprises an inverted measuring space and wherein the transfer tube outlet opening is positioned inside the inverted measuring space.

11. An assembly composes of the sensing device according to any one of more of the claims 1-8 and the preparation apparatus according to claim 9 or 10.

12. A method for forming a sensing device for detecting an analyte according to any one or more of the claims 1-8, wherein the sensing device at least comprises a substrate having a substrate surface formulated to bind to the analyte, the method comprising at least the steps of: i) providing the substrate having a substrate surface containing polycrystalline graphite or polycrystalline diamond; ii) oxidizing the polycrystalline graphite or polycrystalline diamond containing substrate surface through immersion in a 3 / 1 mixture of sulphuric acid (H2SO4) and hydrogen peroxide (H2O2) ; iii) heating the mixture to 60 °C for 1 h; iv) rinsing the oxidized substrate with water and methanol; v) drying the oxidized substrate in an oven for 30 minutes at 65 °C; vi) immersing the oxidized surface in a solution of APTES in toluene (5% v / v); vii) heating the immersed oxidized surface to 75 °C for 2 h; viii) rinsing the oxidized surface with water and methanol and drying the oxidized surface in an oven for 30 minutes at 65 °C.

13. A method for detecting an analyte in a sample of air, in particular in a sample of respiratory air using the preparation apparatus according to claim 9 or 10 and the sensing device according to any one or more of the claims 1 to 8, the method comprising the steps of: a) guiding an air flow containing the analyte through a certain amount of liquid sample medium contained in the first holder of the preparation apparatus, allowing the analyte to be contained in the certain amount of liquid sample medium whilst measuring the amount of air flowing through the certain amount of liquid sample medium in the second holder of the preparation apparatus; b) passing the certain amount of liquid sample medium containing the analyte through the sensing chamber and over the substrate surface formulated to bind to the analyte; c) measuring an impedance across the substrate; and d) calculating an amount of the analyte bound to the substrate surface based at least in part on a change in impedance being measured.

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