WO2019045647A1

WO2019045647A1 - Sensing device, methods and uses thereof

Info

Publication number: WO2019045647A1
Application number: PCT/SG2018/050435
Authority: WO
Inventors: Vignesh Suresh; Bera LAKSHMI KANTA; Karen Siew Ling CHONG; Yeong Yuh LEE; Qunya Ong
Original assignee: Agency for Science Technology and Research Singapore
Current assignee: Agency for Science Technology and Research Singapore
Priority date: 2017-08-31
Filing date: 2018-08-29
Publication date: 2019-03-07
Anticipated expiration: 2020-02-29

Abstract

The invention relates to a sensing device for quantitatively measuring a concentration of an analyte. The device comprises a hydrophilic substrate having a surface that is partially impregnated with a hydrophobic material, a non- impregnated portion of the first surface defining at least one reaction zone. An enzyme is located in the reaction zone for converting the analyte into at least a charged species. The sensing device also comprises a pair of electrode spaced apart and in contact with the reaction zone for conducting a current generated by diffusion of the charged species so as to quantitatively measure the concentration of the analyte. In a preferred embodiment the hydrophilic substrate is paper, the hydrophobic material is wax and the enzyme is urease.

Description

SENSING DEVICE, METHODS AND USES THEREOF

FIELD

The present disclosure relates generally to a sensing device, methods and uses thereof. In particular, the present disclosure relates to a sensing device for quantitatively measuring an analyte, the related method of fabrication and its uses.

BACKGROUND

Recent advances in microfluidics make use of different material substrates such as polymers, paper and overhead transparency sheets that enable controlled flow of fluid through micro channels and thus, are not limited to device channels conventionally made using polydimethylsiloxane (PDMS). In the last few years, there has been a tremendous growth in fluid flow dynamics, process optimization, analyte detection, device fabrication and electronics integration for microfluidics that have enabled the use of microfluidics in healthcare, forensic analysis and environmental monitoring applications. However, these devices have been limited to qualitative measurements.

A problem with microfluidic devices is that accurate control is required and minor perturbation will throw the system off. For example, because of the micrometric dimensions of the tubes and channels, air bubbles in the devices can be very difficult to remove and be very detrimental for the device. Further, small particulates can cause blockage in the channels.

Additionally, current technologies can only reach a limit of detection in the μΜ range. For example, a product from Cell Biolabs which uses colorimetry as the method of detection has a limit of detection (LOD) of urea of about 2.5 mM. Devices using electrochemical methods have also been reported in literature. For example, it has been reported that the LOD for glucose using such devices is about 60 μΜ, while in another report, urea, glucose and lactic acid are detected in the μΜ range. Accordingly, there is a need to have a device with better LOD. The limit of detection becomes critical for lower concentrations. Electrochemical sensing typically requires a reference electrode, a counter electrode and a working electrode. The electrode material, surface property and its dimensions (surface area), greatly influence the detection ability (limits) of the electrochemical system. However, it has been found that minute changes in these factors can introduce anomalies, raising concerns regarding the performance outcome and increasing the inaccuracy of these devices. For example, previously reported paper-based microfluidic substrates make use of a three-electrode system (electrochemistry) for sensing. These electrochemical measurements largely depend on the area of the electrodes and thus, may vary from substrate to substrate and introduce inaccuracy. The electrode material also needs to be specifically chosen according to the nature of the analytes as they should be chemically stable.

Thus, there remains a need for low-cost diagnostic assays that are not cumbersome and that can be performed on small sample volumes. There is also a need for a diagnostic assay which is easy to fabricate.

Further, there remains a need for low-cost sensors to detect trace levels of analytes in fluids, for example, for use in human health diagnosis or monitoring, illicit drug use and identification, military and homeland security settings and chemical pollution in the environment.

Accordingly, there is a need to overcome, or at least to alleviate, one or more of the above mentioned problems. SUMMARY OF THE INVENTION

This invention relates to a sensing device for detection of concentration of an analyte such as urea by a non-invasive process. In particular, the invention relates to a sensing device for quantitatively measuring an analyte. The invention is capable, in at least some embodiments, of detecting ultra-low concentration of an analyte. This sensing device can be a paper-based sensor, for electrically measuring ultra-low levels of analyte (for example urea) in the pM range based on a change in current. Such sensing devices are easy to fabricate, can be performed on small sample volumes and can detect trace levels of analytes in fluids. In a first aspect, the present invention relates to a sensing device for quantitatively measuring a concentration of an analyte, comprising:

a) a hydrophilic substrate having a first surface that is partially impregnated with a hydrophobic material, a non-impregnated portion of the first surface defining at least one reaction zone;

b) an enzyme located in the reaction zone for converting the analyte into at least a charged species; and

c) a pair of electrodes spaced apart and in contact with the reaction zone for conducting a current generated by diffusion of the charged species so as to quantitatively measure the concentration of the analyte.

Advantageously, by directly measuring the current generated by diffusion of the charged species in the reaction zone, an analyte with a concentration in a pM to μΜ range can be quantitatively measured. In some embodiments, the reaction zone has a depth of less than 0.25 mm.

Advantageously, this allows an analyte when dissolved or dispersed in small volumes (μΐ. to mL range) to be quantitatively measured. In some embodiments, the sensing device further comprises a colorimetric indicator in the reaction zone for providing a visual indication of the presence and/or the concentration of an analyte.

In a second aspect, the present invention relates to a sensing device for quantitatively measuring a concentration of an analyte, comprising: a) a hydrophilic substrate defining a first surface, the first surface partially impregnated with a hydrophobic material, a non-impregnated portion of the first surface defining at least one reaction zone, an analyte entry port and at least one channel;

b) an enzyme located in the reaction zone for converting the analyte into at least a charged species;

c) a pair of electrodes spaced apart and in contact with the reaction zone for conducting a current generated by diffusion of the charged species so as to quantitatively measure the concentration of the analyte; and

wherein the analyte entry port is in contact with the at least one reaction zone by at least one channel.

The sensing device may comprise a plurality of reaction zones in fluid communication with a single analyte entry port. Advantageously, the properties of a plurality of analytes in a single sample can be measured in a single instance.

In a third aspect, the present invention relates to a method of fabricating a sensing device for quantitatively measuring a concentration of an analyte, comprising the steps of:

a) providing a hydrophilic substrate defining a first surface, the first surface partially impregnated with a hydrophobic material, a non-impregnated portion of the first surface defining at least one reaction zone;

b) locating an enzyme in the reaction zone for converting the analyte into at least a charged species; and

c) spacing a pair of electrodes apart and in contact with the reaction zone for conducting a current generated by diffusion of the charged species so as to quantitatively measure the concentration of the analyte.

In a fourth aspect, the present invention relates to a method of measuring a concentration of an analyte using a sensing device as disclosed herein, comprising the steps of: a) delivering the analyte to the reaction zone;

b) allowing the enzyme to convert the analyte into at least a charged species; and c) measuring a current generated by diffusion of the charged species using the pair of electrodes so as to quantitatively measure the concentration of the analyte.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the present invention will now be described by way of non-limiting example only, with reference to the accompanying drawings in which: Figure 1 illustrates a schematic of a sensing device (such as a paper-based microfluidic device) according to certain embodiments that enables ultra-low detection of properties of analytes (e.g. urea).

Figure 2 illustrates (a) a schematic of a top surface (first surface) of wax printed paper of an exemplary sensing device; (b) a bottom surface (second surface) of the paper; (c, d) a cross section of the device through line 2-2 before and after heating respectively; and (e) a photograph showing the wax on both sides melted and impregnated into the paper.

Figure 3 is a photo of an embodiment of a sensing device subjected to current profile measurement using a semiconductor parameter analyzer.

Figure 4 illustrates current-time profiles of samples with different urea concentrations. The control sample had no pre-loaded enzyme and was tested using 30 μΐ_^ of 1 mM urea as analyte.

Figure 5 illustrates a plot of current measured at t=1000s versus the urea concentration of the samples. The table lists the current measured at varying concentrations of urea.

Figure 6 includes photos of a multi-channel fluidic system fabricated for simultaneous detection of analytes, (a) before use and (b) after use. Figure 7 illustrates probe current (A) versus time (sec) of 30 μΐ_^ samples of sodium lactate with different concentrations measured using the sensing device. The control substrate had no pre-loaded enzyme.

Figure 8 illustrates an example of a multi-channel sensing device for colorimetric detection of various components of various analytes. The color map presents a color scale to quantify the presence, absence or various amounts of components of the analytes.

DETAILED DESCRIPTION

As used herein, the term 'analyte' refers to a substance whose chemical constituent and/or physical property is being identified and measured. The chemical constituent can be a molecule, a biomolecule or an ion. The analyte is dispersed or dissolved in a solvent for it to be detectable and measurable by the sensing device. In this regard, the solvent can be any known solvent medium such as water or biological fluid, such as blood, plasma or urine. Preferentially, the solvent is a polar solvent. Accordingly, the solvent can comprise one or more analytes. For example, if urine is being tested, the analytes may be urea, glucose and/or creatinine. The invention is predicated on the realisation that the current generated by enzymatic breakdown of an analyte and subsequent diffusion of charged reaction products is reflective of the concentration of the analyte. It was further found that the current can be reliably and consistently measured such that an accurate and/or precise reading can be obtained. This output can result in a reading in the pM range; i.e. chemicals can be detected down to picomolar concentrations. Advantageously, the present invention can overcome the limitations of microfluidic devices which are volume dependent. As the sensing devices (such as examples that incorporate a paper based fluidic channel system) are easy and fast to fabricate and versatile, they provide another advantage as they are capable of being disposable point-of-care devices; i.e. low cost substrates (for example paper) can be used for fabricating the sensing device and other low cost material (such as wax) can be used to create the fluidic channels. These are believed to be advantageous for processes in manufacturing and subsequently in commercialisation.

As will be apparent from the disclosure, various embodiments of the invention may comprise three aspects. Firstly, a sensing device can be fabricated by wax printing predetermined designs of a fluidic channel system comprising a reaction zone, entry port, channel and contacts integrated to the reaction zone of a paper substrate. Secondly, the concept of enzyme catalysis can be applied in embodiments of a sensing device; i.e. breakdown of analyte to ions on the sensing device. Thirdly, a readout can be obtained; i.e. measuring the current generated by the released charged species using two-contact set-up, which enables ultra-low detection.

Accordingly, the present invention discloses a sensing device for quantitatively measuring an analyte. Referring now to Figure 1, the sensing device comprises a hydrophilic substrate defining a first surface. The hydrophilic substrate is partially impregnated with a hydrophobic material at the first surface. In this regard, the impregnation of the hydrophobic material in the hydrophilic substrate renders the impregnated regions at the first surface hydrophobic. As the hydrophilic substrate is partially impregnated, a non- impregnated portion or portions at the first surface of the substrate remain hydrophilic. The non-impregnated portion or portions defines at least one reaction zone. An enzyme is located in the reaction zone. The enzyme is placed such that it is suitable for converting the analyte into at least a charged species. Accordingly, the reaction zone refers to the region where an enzymatic reaction occurs. A pair of electrodes (exemplified as silver contacts in Figure 1) is spaced apart and in contact with the reaction zone. In this regard, the electrodes must be touching a portion of the hydrophilic surface such that when in use, it is in contact with the reaction zone.

The pair of electrodes is for conducting a current generated by diffusion of the charged species so as to quantitatively measure the concentration of the analyte. In this regard, the current may be generated when the electrodes are coupled to an external voltage. The current may be generated when the electrodes are coupled to a parameter analyser (Figure 3), though other sources of bias voltage may be used. In this setup, the application of a bias voltage across the pair of electrodes allows for the detection of a current generated due to diffusion of the charged species in the reaction zone for quantitatively measuring the concentration of the analyte.

By measuring the change in current, using this pair of electrodes, that happens when the ions (or charged species) from the analyte are released, the amount of analyte in the sample can be deduced. As would be known to the skilled person, deducing the concentration of the analyte can be performed using a current-concentration plot (for example see Figure 5). Accordingly, there is no need for half cells, or for half-cell reactions to be measured. This allows ultra-low detection of analyte to be acquired from the sensing device.

The hydrophilic substrate of the sensing device can be paper. Various grades of paper can be used. These paper substrates can be porous. For example, various grades of Whatman filter paper can be used. In some embodiments, grade 20 Chr filter paper is used. The skilled person would understand that paper is made from cellulose fibers, and accordingly any cellulose fiber product can be used as the hydrophilic substrate. In this sense, the substrate can be of any shape or size, and does not have to be one dimensional. Fluid transport can occur through the bulk of the hydrophilic region of the paper by capillary action as well as along the first surface. In this regard, the hydrophilic substrate serves to transport the analyte (in a solvent) at least along its first surface, from a first position to another position downstream. The analyte may move together with the solvent front, or behind the solvent front.

The hydrophobic material used to impregnate the hydrophilic substrate (or paper-based porous substrate) may be, but is not limited to, wax, photoresists or polymers. The hydrophobic material can be selected from wax, photoresist, poly dimethyl siloxane (PDMS), hydrophobic resins, polycarbonate, polyethylene, polymethyl methacrylate or polytetrafluoroethylene (PTFE). Accordingly, by varying the amount and type of hydrophobic material, the sensing device can be fabricated with a rigid platform or a flexible platform.

As exemplified in Figure 1, wax is used as the hydrophobic material. Wax partially impregnates the hydrophilic substrate (paper) on at least one surface. Figure 2a illustrates the top surface (also termed as the first surface) of the hydrophilic substrate partially impregnated with wax by patterning the wax on the substrate surface, such that a reaction zone 101 is formed. Other zone and channels (for example 103 and 105) can also be designed and formed. In this regard, the wax forms a boundary for the zones and channels in the substrate.

The hydrophilic substrate also has an opposite surface (i.e., opposite the "top" or patterned surface, also termed as the second surface). The opposite surface (or second surface) can be substantially impregnated with a hydrophobic material. As shown in Figure 2b, the wax can fully impregnate the other surface of the hydrophilic substrate (i.e. bottom surface). In doing so, the whole surface is made hydrophobic. In combination, the partial impregnation of wax on one (top) surface and full impregnation of wax on the other (bottom) surface can create channels and zones with at least three faces surrounded by wax. Advantageously, fully impregnating the bottom surface prevents the analyte, when in use, from seeping through the substrate. The formed channels and zones also retain the analyte in its desired location to effect a good readout. The working volume and sample of analyte required can be reduced (in μΐ) as a result.

In another embodiment, the other surface of the hydrophilic substrate can also be partially impregnated with the hydrophobic material. In this regard, zones and channels can be created on both surfaces or sides of the hydrophilic substrate, thereby allowing the sensing device to be usable on both surfaces/sides. These zones and channels on both surfaces/sides can be in fluid communication with each other or can be separated from each other by a layer of hydrophobic material. The resultant reaction zone (and other zones and channels if present), being made of a hydrophilic substrate surrounded by hydrophobic material, enables capillary flow to be utilized efficiently. This allows the working volume of the analyte to be greatly reduced over a traditional microfluidic channel system.

The enzyme in the reaction zone can be physically bonded to the reaction zone. Alternatively, the enzyme is physically bonded to a paper disc placed in contact with the reaction zone. In this regard, the enzyme is located in the reaction zone to be able to receive the analyte and thereby convert the analyte into at least a charged species.

The enzyme in the reaction zone functions to convert the analyte into at least a charged species. In this regard, the enzyme can digest/breakdown/decompose the analyte or modify the analyte such that a charged species results from its interaction. The charged species can be an ion or a charged molecule. For example, when urea is selected as the analyte, urease is used as the enzyme. The enzymatic breakdown of urea results in NH⁴⁺ ions as the charged species. This charged species is capable of diffusion, which can result in a current being generated and picked up by the electrodes. In an embodiment, the enzyme is selected from urease or lactase. Other analytes can be detected using the sensing device of the present invention, provided that they contribute to enzymatic kinetics; i.e. the enzyme can digest/breakdown the analyte or modify the analyte such that a charged species results from its interaction. A suitable enzyme is accordingly used. In this regard, the analytes which can be detected using the sensing device can be, but not limited to, urea, uric acid, lactate, leukocyte, nitrite, pH, ketone, glucose, creatinine, creatine, pyruvate and β- hydroxybutyrate. The enzymes can be, but are not limited to, urease, carbohydrase, amylase, lactase, and protease. For example, carbohydrase or amylase can be used to break down starch into sugar, lactase can be used to break down lactose into glucose, and proteases can be used to break down proteins into amino acid residues, and hence allow for their detection using the sensing device disclosed herein. More than one enzyme may be located in one reaction zone. In this regard, the analyte may be converted into several charged species. It should also be noted that while enzymes are exemplified, the invention is not limited to enzyme as long as the reaction in the reaction zone generates ions (charged species) that result in a change of current. As mentioned above, the impregnation of the hydrophobic material into the hydrophilic substrate creates zones and channels. The impregnation can further occur on both surface of the hydrophilic substrate. Advantageously, the depth of the zones and channels is reduced to a fraction of the thickness of the substrate, thereby also reducing the effective void volume of the device. This can be done by varying the amount of hydrophobic material used such that the impregnation of the hydrophobic material can be controlled. The reduction in void volume in turn minimizes required sample volume. This is beneficial for a point-of-care diagnostic device.

Accordingly, in an embodiment, the reaction zone has a depth of the same thickness as the hydrophilic substrate. The thickness of the substrate can range from about 0.2 mm to about 2 mm. In another embodiment, the reaction zone has a depth of less than about 0.25 mm. In other embodiments, the reaction zone has a depth of less than about 0.23 mm, about 0.21 mm, about 0.2 mm, about 0.18 mm, about 0.16 mm, about 0.14 mm, about 0.12 mm, about 0.1 mm, about 0.08 mm, or about 0.06 mm.

At least a pair of electrodes is sufficient for the sensing device of the present invention. The electrodes are at least sized and arranged to be contactable with an analysing means for the current to be readable. The analysing means can be a parameter analyser. The electrodes used are also independent of the analyte under examination; i.e. the electrodes are not damaged by the analyte tested. Further, the electrode material, contact material, its properties or the dimension of the electrodes can be varied to suit the sensing device and is independent of the analyte. This is in contrast to previous work, wherein the electrode material has to be chosen according to the nature of the analytes as they should be chemically stable. Depending on the function of the electrode, the electrode material, surface property and its dimensions (surface area), the detection ability of the device is greatly influenced and can pose serious threat to the performance outcome if any of the factors introduce anomaly.

In an embodiment, the electrode is a contact pad. In this regard, the electrode is bonded to the surface of the substrate. The electrode can be made of a conductive metal paste. In some embodiments, the electrode is made of silver paste. Advantageously, by fabricating the electrode as contact pads, good electrical connectivity can be achieved and no specific dimensions are needed. This is in contrast to three-electrode systems, in which the electrode size is critical for the functioning of the system.

The pair of electrodes is capable of conducting a current generated by diffusion of the charged species in the reaction zone (Figure 1 and 3). The current generated can be directly correlated with the concentration of the charged species, which is directly co-related to the analyte. This allows for quantitative measurement of the analyte, and advantageously in the pico-molar (pM) range/concentration. This co-relationship can be plotted in a calibration plot of current vs analyte concentration. As shown in Figure 4, 1 pM of urea can be detected. Figure 5 further illustrates a calibration plot of the current generated vs concentration of urea. It is shown that compared to a control, the detectable range of the sensing device ranges from about 1 pM to about 1 mM. As higher current will be generated from higher concentrations of analyte, it is believed that such higher concentrations of analyte can also be detected (not shown).

For the electrodes to be capable of conducting a current, they are ideally spaced apart at a certain distance. The pair of electrodes can be spaced apart less than about 10 mm from each other. In other embodiments, the electrodes are spaced apart less than about 9 mm, about 8 mm, about 7 mm, about 6 mm, about 5 mm, about 4 mm, about 3 mm or about 2 mm from each other. Advantageously, the spacing is chosen such that the generation of the current from the charged species can be picked up quickly by the electrodes. As the spacing between the electrodes may be required to be wetted such that the electrodes are fluidly communicating via the reaction zone, selection of the spacing helps maintains the fluid connection as well as reduces the amount of fluid required.

The electrodes are also in contact at least with the reaction zone. In some embodiments, the electrodes and the reaction zone are on the same surface of the hydrophilic substrate. This allows the current generated from the charged species in the reaction zone to flow to the electrodes. In some embodiments, the electrodes are screen printed to the reaction zone and/or sensing device using silver paste. This allows the electrode to be electrically communicative with the reaction zone and/or sensing device. The electrodes can be connected to a semiconductor parameter analyser for reading the current signals with respect to time. This is illustrated in Figure 4 and 7. In this regard, the sensing device can further comprise an electrical sensing measurement unit. The electrical sensing measurement unit can be a semiconductor parameter analyser, or a 2, 3 and/or 4 probe terminal current-voltage (I-V), current-time (I-t), voltage-time (V-t) (source measurement units-SMU) measurement system.

For applications that need potential at each of the electrodes to be measured as in a three- electrode system, a third electrode can be integrated onto the device. The sensing device can further comprise a colorimetric indicator in the reaction zone for providing a visual indication of the presence of an analyte. In this regard, detection of the analyte can result in both a colorimetric change and an electrical signal being generated. Referring to Figure 6, the sensing device can depending on the concentration of the analyte, colorimetrically change such that a different color or tone is obtained. In this regard, the change in color or tone can be used to infer, at least visually, the estimated analyte concentration. Figure 8 illustrates the possible color change or color gradient for the different analytes.

The colorimetric indicator can be a pH indicator, specific gravity indicator or colour indicator. For example, Figure 8 shows the colour change that can take place to indicate the presence, absence and quantity (estimation) of, for example, proteins, sugar, and nitrites. In this regard, the colour indicator provides a qualitative reading of the amount of analyte present by providing a colour change. This colour change can be due to, but not limited to, pH change, complexation with a metal center for example iron or redox reactions.

The sensing device can further comprise a barrier material disposed over the reaction zone. This barrier material can be an inert material, which functions to prevent (or at least slow down) evaporation of the solvent. The barrier material also functions to protect the reaction zone from impurities or damage. This barrier material can be an inert tape.

The non-impregnated portion of the surface can further comprise an analyte entry port and at least one channel. The analyte entry port is in contact with the at least one reaction zone by at least one channel. The sensing device can comprise a plurality of reaction zones in fluid communication with a single analyte entry port. In this way, the analyte can be dispensed at the analyte entry port for sensing at the reaction zones. Figure 6 and 8 illustrates the sensing device of the present invention, wherein a plurality of reaction zones is connected to a single analyte entry port. Each reaction zone is connected to the entry port via a channel. As illustrated, four or eight reaction zones can be connected to a single entry port, but is not limited to these examples. More or less reactions zones can be connected, provided that sufficient solvent is provided to the sensing device. In other embodiments the sensing device of the present invention is connected in parallel and sharing the same entry port. In this regard, the channels connecting the reaction zones to the entry port may be of different length or dimensions. Each reaction zone in this sensing device can be set up to detect a different target analyte simultaneously.

Advantageously, the ability to build multiple reaction zones to one entry port allows the sensing device (or paper-based device) to be versatile. This allows sensing of different chemical components of the same analyte, either through colorimetric detection and/or electrical signal, or sensing of different analytes. In this regard, the isolation of the different enzyme kinetics in different reaction zones allows for a clean readout of each individual reaction and subsequently for the reconstruction of the components in the analyte for the full picture. In an embodiment, the entry port and channel independently have a depth of less than about 0.25 mm. In other embodiments, the entry port and channel independently have a depth of less than about 0.23 mm, about 0.21 mm, about 0.2 mm, about 0.18 mm, about 0.16 mm, about 0.14 mm, about 0.12 mm, about 0.1 mm, about 0.08 mm, or about 0.06 mm.

The sensing device of the present invention can further function as a 'switch' . The enzymes causes the breakdown of an analyte into at least a charged species, which can result in a current being conducted to the electrodes. The inventors have found that this can be exploited to make the sensing device act as a switch. When the charged species are released after the enzymatic reaction, the circuit is in a closed loop with the electrodes to allow the flow of electricity. The released charged species can be stopped intentionally (for example, with a slit in the substrate) from diffusing and getting in touch with the electrodes, causing the circuit to be in an open loop and hence, no current will flow. This is advantageous when the reaction has to happen for the color change (colorimetry study) and when no current needs to be measured. Further, it can be useful if the reaction is time delayed; by letting the charged species build up for a certain period of time, a larger current can be detected which can result in a more accurate and/or precise readout.

In an embodiment, the present invention discloses a sensing device for quantitatively measuring a concentration of an analyte, comprising:

a) a paper-based substrate defining a first surface, the first surface partially impregnated with a hydrophobic material, a non-impregnated portion of the first surface defining at least one reaction zone, at least one channel and an analyte entry port, the reaction zone connected to the analyte entry port via the channel;

c) a pair of electrodes spaced apart and in contact with the reaction zone for conducting a current generated by diffusion of the charged species in the reaction zone for quantitatively measuring the concentration of the analyte.

In an embodiment, the present invention discloses a sensing device comprising a paper- based substrate. The paper-based substrate can further be porous. The substrate is selectively impregnated with a hydrophobic material on both the top and bottom surfaces. The top surface of the substrate is selectively patterned with the hydrophobic material such that the non-impregnated portion defines a channel, an entry port and a reaction zone. The bottom surface of the substrate is fully impregnated with the hydrophobic material. With respect to the channel, this creates a channel which is enclosed by the hydrophobic material at at least three faces. An enzyme is immobilized in the reaction zone to convert an analyte into a charged species. Electrodes are printed on the substrate at the reaction zones for conducting a current generated by the diffusion of the charged species and for measurement of the electrical current generated.

a) a paper-based substrate defining a first surface, the first surface partially impregnated with wax, a non-impregnated portion of the first surface defining at least one reaction zone, at least one channel and an analyte entry port, the reaction zone connected to the analyte entry port via the channel;

c) a pair of electrodes spaced apart and in contact with the reaction zone for conducting a current generated by diffusion of the charged species in the reaction zone for quantitatively measuring the concentration of the analyte. In another embodiment, the hydrophilic substrate is paper, the hydrophobic material is wax and at least one enzyme is urease.

Industrial applicability wise, the invention has the potential and can be developed into a hand-held source measurement unit for an integrated portable product (for example, paper fluidic substrate with electronics). Further, these sensing devices, and in particular paper- based substrates, are ideal for point-of-care applications, as they are easily disposable, biodegradable and can be extended to various grades of paper and other analytes contributing to reactions involving release of ions. Multiple configurations can be envisioned as the sensing device (being paper based) can be stacked or even rolled in a tubular manner.

A method of fabricating a sensing device is now described. The sensing device is for quantitatively measuring an analyte. In a first step, a hydrophilic substrate is provided, the substrate defining a first surface. The first surface is partially impregnated with a hydrophobic material, while a non-impregnated portion of the first surface defines at least one reaction zone. At least one enzyme is located in the reaction zone for converting the analyte into a charged species. At least one pair of electrodes is spaced apart and in contact with the reaction zone. The at least one pair of electrodes is capable of conducting a current generated by diffusion of the charged species in the reaction zone for quantitatively measuring the analyte.

The fabrication of the reaction zone is herein described. The hydrophobic material is allowed to impregnate the substrate by firstly printing the design on the first substrate. This design can be a pre-conceptualised design. The hydrophobic material printed substrate is heated to allow the hydrophobic material to diffuse into and impregnate the substrate. This allows the hydrophobic material to flow along the width of the substrate. This creates the reaction zone in the non-impregnated portion, with the impregnated portion acting as a hydrophobic barrier which surrounds the reaction zone. A further heating and/or UV curing step can be added to mix the hydrophobic material in the substrate. The skilled person would understand that the above mentioned fabrication of the reaction zone can be used to fabricate a channel and/or an analyte entry port. This involves making adjustments in the design. In this regard, the pre-conceptualized designs of the sensing device comprises a reaction zone, an entry port and a channel. Accordingly, a channel and an analyte entry port can be further fabricated on the substrate.

The electrode may alternatively be screen printed onto the substrate. A parameter analyser can be further coupled with the electrodes for providing a readout of the current over time. In another embodiment, the sensing device is fabricated using wax as the hydrophobic material. This allows the creation of non-impregnated portion on the surface of the substrate, which is paper.

Advantageously, the fabrication of sensing device is low cost, simple, versatile and scalable. It eliminates the need for complex processes associated with traditional PDMS microfluidic devices. As the sensing device allows fluid flow by capillary action rather than free flow through conduits associated with PDMS channels, it avoid the problem of air traps which can introduce inaccuracies in analyte detection. The sensing device is a non-invasive electronic sensing system for chemical detection using a very cost-effective fabrication strategy. Additionally, colorimetric detection can be implemented together for analyte detection. Colour changes can be calibrated to give an indication of the quantity of analyte present. Multiple analytes in a small volume sample can be simultaneously detected via colorimetric changes by simply tuning the design of the sensing device. The device that couples the advantages of the calorimetric sensing with the detection of change in the ion concentrations will enable ultra-low level detection of analytes at picomolar scale or lesser with enhanced sensitivity.

The disclosed method of fabrication can also be easily extended to build multi fluidic channel system that allows simultaneous detection of ions released during different reaction conditions. Further, paper-based substrate has the advantage of being disposable and environmentally friendly. The low cost of production involved in making this device will also encourage the adoption of this technology in resource- scarce settings.

In use, the analyte is added which flows by capillary action in the reaction zone. If an anlayte entry port is present, the analyte can be added at the entry port. The analyte then flows from the entry port via the channel by capillary action to reach the reaction zone. The generation of charged species by the enzyme in the reaction zone results in a change in current (electrical signal) which can be translated to analyte detection at picoMolar scale or lesser.

In essence, an analyte using a sensing device of the present invention can be measured by firstly delivering the analyte to the reaction zone. The enzyme is then allowed to convert the analyte into a charged species. Subsequently, the current generated is measured with the pair of electrodes, the current being generated by diffusion of the charged species in the reaction zone for quantitatively measuring the analyte.

Those skilled in the art will appreciate that the invention described herein in susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications which fall within the spirit and scope. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates. Certain embodiments of the invention will now be described with reference to the following examples which are intended for the purpose of illustration only and are not intended to limit the scope of the generality hereinbefore described.

EXAMPLES

The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.

Materials and Methods

Filter paper (No. 50, 0.25mm thick, Advantec, Japan) was used in device fabrication. Urease from Canavalia ensiformis (U4002, Sigma Aldrich) was prepared in phosphate buffered saline to a concentration of 50 mg/mL. Urea stock solution was prepared by dissolving urea powder (First Base) in ultrapure water to a working concentration of 1 M. Urea solutions with various lower concentrations were prepared by further dilution of the stock solution with ultrapure water. Urease being unstable at room temperature, the hydrolysis of urea was carried out as soon as the enzymes were immobilized. Fabrication of sensing device (paper-based)

Doubled-sided printing on the filter paper (whatman equivalent 20Chr) was performed using a wax printer (ColorQube 8570, Xerox). Channel designs were printed on the top surface as shown in the schematic illustration (Figure 1), while the bottom side of the paper was fully printed in wax to render it hydrophobic. This, not only prevents leakage of the analyte from the bottom, but also reduces the sample volume that flows through capillary. The wax printed paper substrate was heated on a hot plate at 85°C for 2 min. Wax on both sides of the paper then began to flow and penetrate into the paper. The temperature and timing were critical to determine the depth of penetration of the wax into the paper. The conditions were optimized such that the wax on both sides only penetrated halfway through the paper. Areas with wax printed on both sides would have complete impregnation of wax along the width of the paper. The wax filled regions become hydrophobic while the regions without wax (paper) remain were hydrophilic and form the fluidic channels lined by wax. Figure 1 shows a schematic illustration of the paper-based microfluidic device that enables ultra- low detection of analytes (urea). Analytes enter through the analyte entry port, and flow through the channel via capillary action, before reaching the reaction zone, which is preloaded with enzymes (urease). Electrochemical enzymatic reaction takes place in the reaction zone, generating ions that result in current flow between the electrodes. The electrodes are screen printed using silver paste.

Figure 2a shows a schematic illustrating the top surface of the wax printed paper. The reaction zone and the entry port have diameters of 7 mm, while the interconnecting channel measures 4 mm in length and 1.5 mm in width. Figure 2b shows that the bottom surface of the paper is entirely wax printed to render it hydrophobic and to prevent leakage. Figure 2c and 2d shows cross sections of the device from the electrode-end before and after heating respectively. The wax on both sides has melted and impregnated into the paper as evidenced by the photograph in (e). The proposed simple two-electrode setup will be adequate to detect ultra-low concentration of analytes as it is able to capture even minuscule movement of ions responsible for the closed circuit current. In this work, silver paste was screen printed onto the hydrophilic region of the paper substrate to serve as the metallic contact in the two-electrode configuration (Figure 1). The geometrical dimensions of the wax printed paper are shown in Figure 2. The distance between the two electrodes is 4 mm. The reaction zone and the entry port have diameters of 7 mm.

To immobilize urease in the reaction zone, 7 mm diameter paper discs were pre-loaded with 10 μΐ_^ of 50 mglmL of urease and allowed to dry. The enzyme-loaded discs were then placed on the reaction zone together with a smaller disc of pH indicator cut out from pH indicator strips. The assembly was then secured in place with an inert tape. The reaction zone was connected to the analyte entry port through a small hydrophilic channel 1.5 mm in width and 4 mm in length. Detection of analyte (urea)

30 μΐ aqueous samples of different urea concentrations, 1 mM, ΙμΜ, 1 nM and 1 pM were injected into the entry port and were allowed to reach the reaction zone by capillary action where enzyme catalysis hydrolyzed urea. For each sample, change in pH, as shown by the pH indicator was noted and current profile between the two electrodes was recorded using a semiconductor parameter analyzer (Agilent 4156C). The semiconductor parameter analyzer was set to the following conditions prior to measurement- electrode bias 2V, current compliance 40 mA, time 20 mins. The two point probes were gently pressed onto the silver electrodes. Figure 3 shows current profile measured using a semiconductor parameter analyzer. Paper-based microfluidic devices immobilized with urease were tested with samples containing ImM, ΙμΜ, Inm and lpM of urea. The measurement was started without introduction of any sample. After 100s, 30 μΐ of sample was immediately injected into the entry port and allowed to reach the enzyme-loaded reaction zone by capillary action. The change in current with respect to time was measured. A control run was carried out using a substrate that was not loaded with urease. 30 μΐ of 1 mM urea was dispensed through the entry port and the above-mentioned measurements were carried out.

Results

An electrochemical paper-based device, comprising of an entry port, a fluidic channel, a reaction zone and two electrodes, was fabricated (Figure 1). Samples containing urea were injected into the entry port and the fluid flowed through the channel via capillary action, before reaching the reaction zone, which was preloaded with urease. In the course of enzymatic hydrolysis of urea, ammonium carbonate was produced, which dissociated into several ions, and the reaction could be monitored by measuring the current flow between the electrodes.

Figure 4 shows current-time profile of samples with different urea concentrations. The control sample had no pre-loaded enzyme and was tested using 30μ1 of ImM urea as analyte. It could be observed from the current-time plot in Figure 4 that there was a sudden sharp increase in current when the electrodes came in contact with the liquid front. Urea in aqueous solution is generally electrolytically conducting. As the liquid front reached the reaction zone, urea was catalyzed by urease into releasing ammonium ions. Ions in the liquid closed the circuit between the electrodes and the current increased as the enzymatic reaction took place. Since, the fluid is transported by capillary action and as long as the fluid saturates the hydrophilic channels, a contact with the electrodes is established that captures the minuscule change in conductivity. The measured current, therefore; does not arise from bulk fluid flow as observed in traditional microfluidic channel where fluid flows in bulk through the conduit. The current plateaued over time and remained stable as long as the paper remained wet. Evaporation was mitigated by carefully covering the substrate with inert tape.

Figure 5 shows plot of current measured at t=1000s versus the urea concentration of the samples. The current measured increased linearly with analyte concentration. The table lists the current measured at varying concentrations of urea. Results show that lpM can be significantly be detected. The limit of detection and range of urea sensors can be customized based on the requirements. While sensors for measuring urea in urine, blood and serum need to be able to pick up concentrations ranging from a few micromolar to several millimolar, sensors for trace urea detection need to have ultralow detection limit ranging from a few nanomolar to picomolar. Thus, the technology as a whole can be extended to various grades of paper and other analytes contributing to enzyme kinetics.

The electrochemical paper-based fluidics system can be improvised for simultaneous detection of analytes by adding multiple reaction zones that share a common entry port. Response of the injected analyte under different reaction conditions can be studied by varying parameters such as enzyme concentration, injection volume and pH.

The versatility of the system is demonstrated in Figure 6. Figure 6 shows photos of a multichannel fluidic system fabricated for simultaneous detection of analytes. The as-prepared substrates with preloaded urease of concentration 50mg/ml and pH marker as seen in (a). Reaction zones in the device in Figure 6a were immobilized with urease of varying volumes 10 ul, 20 ul, 20 μΐ and 0 μΐ (control), from left to right. A pH marker was attached to each reaction zone. The pH indicator discs initially appeared yellow before sample injection. Figure 6b shows the multi-device after the test was performed using 30 μΐ of 1M urea in water dispensed in the common port of entry. All, except the control with no urease, changed color after 30 μΐ of 1M urea was introduced in the common port of entry (Figure 6b). The first three reaction zones (1, 2, 3) shows a change in color. This suggested that the release of ions was due to the catalysis of urea by urease, indicating that enzymatic reaction had taken place. The control reaction zone (4) had no enzyme, thus no color change and no enzymatic reaction occurred and pH remained unchanged.

The said invention is not limited for specific chemical detection as demonstrated above; rather it is versatile for detection of analytes that favour enzymatic reactions. The method has been tested to detect another enzymatic reactive chemical such as sodium lactate as demonstrated in Figure 7. Figure 7 shows probe current (I) versus time (t) characteristics during enzymatic reaction with different dilution level of sodium lactate. The control substrate had no pre-loaded enzyme. The total volume of sodium lactate used for all the experiments are 30 μΐ. The results demonstrate that the sensing device can be used with other enzymes for the detection of analytes through the conversion of analytes to charged species and the capability of the invented technique to detect analytes of concentrations in the picomolar scale. In this example, the enzyme used is lactate oxidase. This enzyme oxidizes lactate in the presence of oxygen to pyruvate and hydrogen peroxide. A further reaction of the resulting hydrogen peroxide can occur as aqueous analyte continues to flow by capillary action, thus generating ions such as H₃0⁺ and 0₂H^~.

The demonstrated paper-based microfluidic device made use of the laboratory scale parameter analyzer to measure, for example, current, time and voltage characteristics. As an extension, the proposed paper-based non-invasive electrical sensing device can be further equipped to encompass a portable source measurement unit to analyze the current, voltage, time characteristics similar but not limited to point-of-care device kits. The microfluidic channel design would consist of pre-conceptualized geometry according to the analyte(s) that need to be characterized. Figure 8 illustrates a multi-channel sensor device for detecting various analytes and physiological conditions such as leukocytes, nitrites, protein, pH, blood, specific gravity, ketone and glucose. The color map presents a color scale of each analyte or condition in the absence or presence of various amounts of analyte or condition. A pair of electrodes can be further provided at each reaction zone for conducting a current generated by diffusion of the charged species in the reaction zone such that the concentration of an analyte can be quantitatively measured. In this regard, the sensing device is capable of measuring multiple analytes, and can comprise an electrical measurement unit and read-out display unit. The sensing device can thus be used for quantitative (current) and qualitative (colour) measurements simultaneously. Advantageously, the concurrent measurements provide a feedback so that a user can check to make sure that both readings for a given analyte approximately correlate to each other. Further, the qualitative measurement provides for a quick analysis of the various analytes, so that the user can subsequently choose to focus his attention on the analytes that are of more importance.

Claims

CLAIMS DEFINING THE INVENTION

1. A sensing device for quantitatively measuring a concentration of an analyte, comprising:

c) a pair of electrodes spaced apart and in contact with the reaction zone for conducting a current generated by diffusion of the charged species in the reaction zone so as to quantitatively measure the concentration of the analyte.

2. The sensing device of claim 1, wherein the hydrophobic material is selected from wax, photoresist, poly dimethyl siloxane (PDMS), hydrophobic resins, polycarbonate, polyethylene, polytetrafluoroethylene (PTFE).

3. The sensing device of claim 1 or 2, wherein the hydrophilic substrate is paper.

4. The sensing device of any of claims 1 to 3, wherein the enzyme is physically bonded to the reaction zone or physically bonded to a disc or sheet of hydrophilic material placed in contact with the reaction zone.

5. The sensing device of any of claims 1 to 4, wherein the enzyme is selected from urease or lactase, carbohydrase, amylase and protease.

6. The sensing device of any of claims 1 to 5, wherein the reaction zone has a depth of less than 0.25 mm.

7. The sensing device of any of claims 1 to 6, wherein the pair of electrodes are spaced apart less than about 10 mm from each other.

8. The sensing device of any of claims 1 to 7, further comprising a colorimetric indicator in the reaction zone for providing a visual indication of the presence and/or concentration of an analyte.

9. The sensing device of claim 8, wherein the colorimetric indicator is a pH indicator, specific gravity indicator or colour indicator.

10. The sensing device of any of claims 1 to 9, further comprising a barrier material disposed over the reaction zone.

11. The sensing device of any of claims 1 to 10, the non-impregnated portion of the first surface further comprising an analyte entry port and at least one channel, the analyte entry port being in contact with the at least one reaction zone via at least one channel.

12. The sensing device of claim 11, comprising a plurality of reaction zones in fluid communication with a single analyte entry port via respective channels.

13. The sensing device of any of claims 1 to 12, wherein the hydrophilic substrate has a second surface opposite the first surface, the second surface being substantially impregnated with a hydrophobic material.

14. The sensing device of any of claims 1 to 13, wherein the hydrophilic substrate is paper, the hydrophobic material is wax and at least one enzyme is urease.

15. A method of fabricating a sensing device for quantitatively measuring a concentration of an analyte, comprising the steps of:

a) providing a hydrophilic substrate defining a first surface, the first surface partially impregnated with a hydrophobic material, a non-impregnated portion of the first surface defining at least one reaction zone; b) locating an enzyme in the reaction zone for converting the analyte into at least a charged species; and

c) spacing a pair of electrodes apart and in contact with the reaction zone for conducting a current generated by diffusion of the charged species in the reaction zone so as to quantitatively measure the concentration of the analyte.

16. A method of measuring a concentration of an analyte using a sensing device of any of claims 1 to 14, comprising the steps of:

a) delivering the analyte to the reaction zone;