GB2616016A

GB2616016A - Integrated microfluidic test strip

Info

Publication number: GB2616016A
Application number: GB2202485.5A
Authority: GB
Inventors: White Richard; Hurth Cedric
Original assignee: Mint Diagnostics Ltd
Current assignee: Mint Diagnostics Ltd
Priority date: 2022-02-23
Filing date: 2022-02-23
Publication date: 2023-08-30
Also published as: WO2023161619A1; GB202202485D0

Abstract

Test device [10, Fig. 1] comprising: at least one reagent port [22, Fig 2a] accepting at least one reagent under pressure from a reagent pouch; a sample port 21 for a bodily fluid sample; a common port 23 comprising an output and at least one input, each input coupled to one reagent port by a reagent channel 24, the output of the common port joined to the sample port by a transfer channel [41, Fig 4a]; and at least one reaction chamber 25. The reaction chamber comprises a plurality of electrodes to perform at least part of a biosensing test of a substrate solution comprising the bodily fluid sample and the reagent and has one or more bioreceptors for binding to a target analyte. Each reaction chamber is coupled to the sample port by a retention channel. A top surface of the common port comprises a gas-permeable membrane configured to allow gas to pass through but prevent fluid.

Description

Integrated Microfluidic Test Strip

FIELD OF THE INVENTION

The present invention generally relates to a test device for bodily fluids, in particular for saliva.

BACKGROUND TO THE INVENTION

Point-of-care devices need to remove user variability and be as simple as possible to be reliable even for very inexperienced users. This means that the addition of reagents necessary to carry out a specific biochemical test would preferably not be added by the user using, for instance, a micropipette as it is done in laboratories. In more detail, current methods for automating biomedical assays include: coupling to external pumps like compact micro-pumps that can displace small volumes within 5-100 pL with high accuracy but come at a cost and need complex integration efforts embedding the reagents in a lyophilized form and re-dissolving them, but this often affects their activity and functionality manually introducing reagents with a dispensing device like a micropipette, but this involves complex manipulation by the user For use in understanding the present invention, the following disclosures are referred to: * Blister pack integration of reagents is known including C. Moche et al., DE102016122056B4, "Microfluidic system for the intake, delivery, and movement of fluids", 2021.

* Smith S. et a!, Microfluid Nanofluid (2016), 20:163 * GB 2104622.2 (awaiting publication): This document discloses a bodily fluid collection apparatus for collecting and storing a predetermined volume of bodily fluid such as saliva, the device comprises a collection tool comprising at least one internal channel or cavity extending from a first end to a second end of the collection tool, the first end of the tool for receiving a bodily fluid directly from a person; a storage vessel configured to couple to and decouple from the second end of the collection tool at one end of the storage vessel; a fluid displacement means configured to fit the first end of the collection tool such that a movement of the fluid displacement means causes air displacement to transfer bodily fluid from inside the at least one internal channel or cavity of the collection tool into the storage vessel; and a vent system configured to allow air to escape from the channel or cavity during the fluid displacement. In an embodiment, the storage vessel comprises a removable seal at an end opposite the end that is configured to couple to the collection tool. Further, the seal may comprise a plug and the storage vessel may be configured to couple with a test device such that when the storage vessel is coupled to the test device, the plug opens to allow the bodily fluid to move from the storage vessel to the test device. In an embodiment, the storage vessel, when not coupled to the collection tool, is configured to be coupleable to a storage cap. If the collected bodily fluid does not need to be used in test(s) straight away, it can be stored separate from the collection tool. In an embodiment, the fluid displacement means comprises a plunger. In an embodiment, the at least one internal channel or cavity comprises a capillary channel having a hydrophilic surface, eg material, coating or adhesive tape. Furthermore, the hydrophilic surface of the capillary channel preferably extends from the first end to a seal, eg plug, at the second end of the collection tool.

* GB 2003979.8 * Zimmermann M. et al, Lab Chip (2007), 7: 119 * Vasilakis N. et at Microfluid Nanofluid (2017), 21:103 Therefore, there is a need in the field of bodily fluid collection devices for improvements such as, e.g. reducing user variability, and reducing the requirements for user interaction such as pipetting reagents.

SUMMARY

According to a first aspect of the invention, there is provided a test device for bodily fluids comprising: at least one reagent port configured to accept at least one reagent under pressure from a reagent pouch; a sample port configured to accept a bodily fluid sample; a common port, comprising an output and at least one input, each at least one input of the common port coupled to one reagent port of the at least one reagent port by a reagent channel, the output of the common port coupled to the sample port by a transfer channel; and at least one reaction chamber comprising a plurality of electrodes to perform at least part of a biosensing test of a substrate solution, the substrate solution comprising the bodily fluid sample and the at least one reagent, the reaction chamber functionalized with one or more bioreceptors for binding to a target analyte, each reaction chamber of the at least one reaction chamber coupled to the sample port by a retention channel; and wherein a top surface of the common port comprises a gas-permeable membrane configured to allow gas to pass through but prevent fluid.

Advantageously, the membrane of the common port absorbs pressure, such as burst pressure, that is created when the reagent is inserted into the reagent port. If the pressure was not absorbed, then the pressure would cause the fluidic features of the test device to be flooded with reagent. Furthermore, by including a membrane that does not allow fluid to pass, it ensures that the reagent is confined within the test device and does not escape to the outside environment.

In an embodiment, the test device is a microfluidic device.

In an embodiment, the at least one input of the common port is angled in a direction towards the outlet of the common port to facilitate rapid draining of the common port.

Advantageously, this feature facilitates rapid draining of the common port. Otherwise it would take a lot longer to drain.

In an embodiment, the common port has an asymmetric geometry about an axis passing through the centre of the outlet of the common port, wherein the common port comprises an opposing wall opposite an inlet wall comprising at least one inlet of the common port, the inlet coupled to the reagent channel, wherein the opposing wall extends from the outlet at a lower angle that the inlet wall.

Advantageously, the opposing wall reduces the velocity of the reagent which hits it when it enters the common port from the reagent channel.

Advantageously, this feature favours rapid drainage of the reagent into the outlet of the common port on the closest to inlets and prevents turbulent flow on the side opposite the inlets to reduce bubble formation. This feature redistributes the jet inlet stream to the entire surface of the port. It can also allow increasing the port capacity.

In an embodiment, the common port has a capacity greater than a maximum released volume from each reagent pouch.

Advantageously, this reduces the possibility of flooding the features of the test device as the reagent is added under pressure.

In an embodiment, a capacity of the at least one reagent channel is configured to be equal to a volume of unneeded reagent from the reagent pouch.

Advantageously, this feature allows the reagent channel to define/store a waste or unused volume of reagent that is not required for the assay.

In an embodiment, an inner surface of the reagent channel comprises a hydrophilic material, wherein inner surfaces of the common port do not comprise a hydrophilic material.

Advantageously, this assists in causing an amount of reagent to be stored in the reagent channel.

In an embodiment, a cross-sectional area of the retention channel is lower than a cross-sectional area of the inlet of the at least one reaction chamber.

Advantageously, this prevent fluids and reagents from moving from the reaction chambers back towards the common port which may result in unwanted mixing of reagents.

In an embodiment, a cross sectional area of the transfer channel is smaller than a cross sectional area of the at least one reagent channel and a cross sectional area of the inlet of chamber of the common port.

Advantageously, this ensures that the capillary pressure is highest at the outlet of the common port and ensures that bubbles formed in the common port 23 do not enter the capillary-driven section of the device downstream.

In an embodiment, the reagent port further comprises at least one piercing structure configured to burst the reagent pouch when the reagent pouch is coupled with the reagent port and an external pressure is applied to the reagent pouch.

In an embodiment, the sample port comprises a conical-like section and a through-hole, wherein the conical-like section comprises a surface that forms an angle of at least 65 ° with an axis passing through the centre of the through-hole in the direction of fluid flow.

In an embodiment, the sample port further comprises a cylindrical-like part, the conical-like section located between the cylindrical-like part and the through-hole.

In an embodiment, an inner surface of the sample port comprises a hydrophilic material.

In an embodiment, the at least one reaction chamber comprises at least two reaction chambers, the reaction chambers coupled together by a curve channel, wherein the curve channel comprises at least one turn.

Advantageously, this provides a higher hydraulic resistance to limit the contamination between samples measured in each reaction chamber.

In an embodiment, the at least one reaction chamber comprises at least four reaction chambers.

In an embodiment, a capillary pressure differential from the reagent channel to the common port prevents flow from the common port to the reagent channel.

In an embodiment, the at least one reagent channels are separated by a distance such that there are no leakage paths between the reagent channels.

In an embodiment, the microfluidic test device further comprises a capillary pump, the capillary pump coupled to the at least one reaction chamber.

In an embodiment, the sample port contains a protruding structure configured to pierce a sample collection tool.

According to a second aspect of the invention, there is provided a fluid sample test sys-tem comprising the microfluidic test device detailed above and a reader device for controlling at least one of the test electrodes to perform the at least part of the biosensing test, and to output a result of the biosensing test.

According to a third aspect of the invention, there is a use of the microfluidic test device detailed above or the test system detailed above, to perform an ELISA or ELONA test.

In an embodiment, the use comprises: (i) receiving a bodily fluid sample in the sample port; and (ii) receiving a reagent in the at least one reagent port. BRIEF DESCRIPTION OF THE DRAWINGS For a better understanding of the invention and to show how the same may be carried into effect, reference will now be made, by way of example, to the accompanying drawings, in which: Fig. 1 the structure of a microfluidic test device according to an embodiment; Fig. 2A illustrates a top surface of the fluidic channel structure layer1 when the test device is in use according to an embodiment; Fig. 2B illustrates a bottom surface of the fluidic channel structure layer when the test device is in use according to an embodiment; Fig. 3A illustrates a 3D CAD rendering of the saliva port according to an embodiment; Fig. 3B illustrates a cross-section around the major diameter of the saliva port according to an embodiment; Fig. 4A shows a 3D rendering of the channel structure in the bonding layer; Fig. 43 illustrates an overlap of the bonding layer and the features on the bottom surface of the moulded fluidic channel structure layer; Fig. 5A illustrates a reagent port in relation with off-the-shelf reagent pouches according to an embodiment; Fig. 53 illustrates a portion of a reagent port according to an embodiment; Fig. 5C shows a schematic of the release of the reagent fluid according to an embodiment; Fig. 6A illustrates the configuration of the reagent channels from the reagent ports to the common port in a transparency model according to an embodiment; Fig. 68 illustrates the interface between the reagent channels and the common port according to an embodiment.

Fig. 7A illustrates the structure of the common port according to an embodiment.

Fig. 73 illustrates use of the bonding layer to structure the connection between the reagent channels and the common port according to an embodiment.

Fig. 8 illustrates the different steps of the injection of reagent in the common port via the reagent channels and the subsequent draining into the outlet channel according to an embodiment.

Fig. 9 illustrates the fluidic structures in the bonding layer allowing reagents to move from the common port to the reaction chambers according to an embodiment; Fig 10A shows a footprint of the capillary pump according to an embodiment; Fig. 108 illustrates details of the diamond pillars of the capillary pump according to an embodiment; Fig. 11A -11D illustrates the electrode (sensor) layer according to an embodiment; Fig. 12 illustrates a simplified flow diagram of the order of the steps preferably all of which are implemented according to an embodiment; Fig.13 illustrates a reader device according to an embodiment; Fig.14A shows a top view of the microfluidic test device with the reagent pouches according to an embodiment; Fig.14B shows a bottom view of the microfluidic test device with the reagent pouches according to an embodiment; Fig. 15A shows an overlay of the moulded fluidic channel structure layer and the bonding layer according to an embodiment; and Fig. 15B shows cross sections corresponding to Fig. 15A.

DETAILED DESCRIPTION OF EMBODIMENTS

Additionally or alternatively to reducing user variability and/or being simple for the user, a point-of-care device preferably does not use any external pressure or vacuum sources so that it can function in a variety of settings, including environments with limited resources or outside of a laboratory. To remove the need for an external source to move the fluids (reagents) to their targets, our preferred approach is to rely on capillary forces.

To ensure reliable addition, reagents could be injected at high pressures. However, if the pressure is not contained, this will perturb the evolution of fluids by capillarity in the analytical part of the device. A preferred embodiment of our invention couples the high-pressure and low-pressure (capillarity) sub-sections of a functioning point-of-care device.

An embodiment provides for the automated measurement of the hormone content of saliva samples using embedded reagents. The reagents are contained with collapsible compartments that can be pressed manually by inexperienced users or automatically by

B

inserting it into a mechanical actuator. Aside from the actuator, the device may complete the workflow without any intervention form external sources relying on capillarity to guide the fluid within the device.

Fig. 1 illustrates the structure of a microfluidic test device 10 according to an embodiment. The assembled test device 10 comprises three layers: a moulded fluidic channel structure layer 11, a bonding layer 12, and an electrode layer 13. These layers are described further below.

The layers 11 12 13 shown in Fig. 1, may be assembled together so that the bonding layer 12 is sandwiched in between the fluidic channel structure layer 11 and the electrode layer 13. In an alternative embodiment the layers may be assembled together by screwing or otherwise joining the layers together so that the bonding layer is sandwiched in between the fluidic channel structure layer 11 and the electrode layer 13. In other embodiments, thermal or solvent bonding may be used.

While Fig. 1 and the description below describes three distinct layers that are assembled together to create the microfluidic test device 10, the microfluidic test device 10 may also be created from a single piece of moulded plastic.

The fluidic channel structure layer 11 is a plastic layer that contains most of the fluidic features and is the support for the surface chemistry that enables the assay. Fig. 2A illustrates a top surface of the fluidic channel structure layer 11 when the test device 10 is in use. The top surface of the fluidic channel structure layer 11 is the user interface side of the test device 10. Fig. 2B illustrates a bottom surface of the fluidic channel structure layer 11 when the test device 10 is in use.

Referring to Fig. 2A and Fig. 2B, the fluidic features of the fluidic channel structure layer 11 include a saliva port 21 and at least one reagent port 22. The fluidic channel structure layer 11 also comprises a common port 23. In the embodiment, the common port 23 comprises an opening in the fluidic channel structure layer 11 to which a membrane may be attached to define the top surface of the common port 23. The common port is described in further detail below.

On the bottom surface of the fluidic channel structure layer 11, there is at least one reagent channel 24. Each of the reagent channels 24 connects one reagent port 22 to the common port 23, so that any fluid that is entered into the reagent port 22 moves through the reagent channel 24 to the common port 23.

On the bottom surface of the fluidic channel structure layer 11, there is at least one reaction chamber 25 in which the assay is performed, and a capillary pump 26 coupled to the at least one reaction chamber.

The bonding layer 12 is a double-side adhesive layer made of one or more pressure-activated adhesives. The bonding layer 12 may comprise of a laminate with an adhesive material or substance applied to the top side and an adhesive material of adhesive or substance applied to the bottom side. The bonding layer 12 functions to join the fluidic channel structure layer 11 and the electrode layer 13 together.

The bonding layer 12 also provides a hydrophilic surface to some of the fluidic features defined in surface of the fluidic channel structure layer 11. The bonding layer 12 also comprises a number of features cut out of the layer. These cut-outs further define the common port 23, as well as defining a transfer channel 41, a retention channel 42, and a curve channel 44 which overlaps the reaction chambers 25 (see Fig. 4A).

In an alternative embodiment, the curve channel 44 is defined in the fluidic channel structure layer 11.

The electrode layer 23 comprises the electrodes of sensors to enable measurements to be made in the assay. The electrode layer 23 comprises gold tracks deposited on or ablated from a plastic support with a total thickness of 0.2 mm in the preferred embodiment.

The following technical description will follow the path followed by a fluid sample during an assay performed in the overall assembled test device 10 of Fig. 1.

The first step a user may carry out is to load the sample (e.g., saliva) into the saliva port 21. To achieve this the user uses a sample collection tool, pipette or other means, as described below. While saliva is referred to throughout the rest of the description, bodily fluids other than saliva may be loaded instead, such as urine, sweat, blood etc. The saliva port 21 of Fig. 2A is shown in further detail in Fig. 3A and Fig. 3B. Fig. 3A shows a 3D CAD rendering of the saliva port 21 on the edge of the fluidic channel structure layer 11 of the assembled test device 10. Fig. 3B illustrates a cross-section around the major diameter of the saliva port 21.

The saliva port 21 consists of an opening with several sections: i) a cylindrical -like part 31 at the top of the fluidic channel structure layer 11 to create volume, ii) a conical-like section 32 in the middle of the fluidic channel structure layer 21 with a bottom angle of at least 65°, and iii) a through hole 33 connecting the conical-like section 32 to a bottom face of the fluidic channel structure layer 11 to connect to the transfer channel 41, and the retention channel 42 on the bonding layer 12.

In a preferred embodiment, the fluidic features are created in a plastic material using injection moulding, but laser ablation for suitable materials including acrylic sheets may also be used. The nature of the plastic material used to mould the fluidic channel structure layer 11 with the fluidic features can vary and is chosen to optimize the efficiency of the surface chemistry preferred to carry out the intended assay. Polystyrene is a preferred material for antibody deposition.

However, polystyrene is hydrophobic in nature. Therefore, in a preferred embodiment, a blocking treatment (e.g., using bovine serum albumin (BSA), PVP, PEG, PVA, fish gelatin, casein, milk, or mixes of these substances) is applied to the surface of the polystyrene to make the polystyrene surface more hydrophilic and guarantee the fluid deposited in the saliva port 21 can cross through the through hole 23, so that the transfer channel 41 and the retention channel 42 on the bonding layer 12 guide the fluid sample to the chemically-derivatized surfaces of the reaction chambers 25.

In an embodiment, the length of the through hole 33 connecting both the fluidic features on the top of the fluidic channel structure layer 11 to those on the bottom of the fluidic channel structure layer 11 is minimized (typically 0.3 mm) to decrease the resistance to the propagation of the sample fluid on a potentially hydrophobic surface.

In a preferred embodiment, the conical-like section 33 is not exactly conical so that the centre of the through hole at the bottom of the conical-like section is offset from a centre of cylindrical-like section 31. In other words, the conical-like section is not symmetrical about a line extending from a centre. In the presented embodiment, this is a preferred layout so that this feature does not interfere with the casing of the reader device in which the test device will be inserted.

The angle of the conical-like section 32 of the saliva port 24 is also a feature that promotes the wetting of the through hole 33. This can help promote fluid flow into the through hole 33 even though the material from which the through hole 33 is made is hydrophobic such as polystyrene.

The extra pressure to force the saliva sample into the hydrophobic hole through hole 33 is given by the difference between the hydrostatic pressure of the column of fluid (height: h) and the capillary pressure at the top of the saliva hole (radius: r): AP = Psauvagh 2ysauvacos(0) where n rsaliva and v saliva are the density and interfacial tension of the saliva sample, respectively, and B is the contact angle between the saliva sample and the hole surface.

Using the parameters of the preferred embodiment, the maximum contact angle for which the fluid enters the through hole 33 is 89° (AP > 0). In a preferred embodiment, the conical section 32 of the saliva port 21 features an angle of 73° between the bottom of the conical section 32 and the direction of the through hole 33 connecting it to the fluidic features on the other side of the fluidic channel structure layer 11, as illustrated in Fig. 33. By using an angle of 73° for the conical section 32, the fluid is forced to wet the entrance of the through hole 33 and transfer to the fluidic circuit on the other side of the fluidic channel structure layer 11.

In a preferred embodiment, the saliva port 21 contains a protruding structure that pierces a sample collection tool (e.g., for saliva, such as that in GB 2104622.2) allowing bodily fluids stored in the tool to move into the saliva port 21. The sloped bottom of the saliva port 21 (sloped conical-like section 33) remains to promote the wetting of the through hole 33 transferring the fluid to the fluidic circuit on the other face of the fluidic channel structure layer 11. Alternatively, the saliva port 21 does not contain a protrusion and would allow for dispensed addition of saliva using either a dropper or a micropipette or another apparatus capable of dispensing a volume of between 10 and 40 microlitres.

Once the saliva sample has transited out of the saliva port 21 via the through hole 33, it enters the channel structure defined in the bonding layer 12.

Fig. 4A shows a 3D rendering of the channel structure in the bonding layer 12. The channel structure comprises a transfer channel 41, a retention channel 42, a common port 23, and a curve channel 44. The channel structure may be defined using laser cutting, but other techniques may be applied. The circular area 45 represents the maximum diameter of the saliva port 21, i.e. the diameter at the top of the saliva port 21. The circular area 46 represents the through hole 33 at the bottom of the saliva port 21.

The transfer channel 41 couples the outlet 47 of the common port 23 to through hole 33 of the saliva port 21. The width of the transfer channel 41 around the through hole 33 is slightly lower than the through hole 33 diameter to ensure the presence of a strong side flow from the exposed hydrophilic adhesive that guarantees that the flow is not interrupted by the presence of a bubble inside the through hole 33 of the saliva port 21.

The retention channel 42 couples fluid between the through hole 33 of the saliva port 21 and the at least one reaction chamber 25. The retention channel 42 prevents the back-flow of the fluid to the common port 23, which would affect the sample incubation in the reaction chamber. (Instances of 'prevent' herein generally mean to reduce or fully prevent).

The curve channel 44 joins each of the at least one reaction chambers 25 in the same unidirectional fluidic pathway to the capillary pump 26. The turns between the at least one reaction chambers 25 provide a higher hydraulic resistance to limit the contamination between samples measured in each reaction chamber 25. The angle of the turns in the curve channel define the hydraulic resistance between the chambers which may be needed to reduce cross-talking of reagents and analytes from one chamber to the next. In Fig. 4A, the turns are shown to be 90 degrees, but other values of turn angle can be used. In a further embodiment, all turns in the curve channel need not be of the same angle.

The hydrophilic adhesive of the bonding layer 12 is recessed along both edges of each reaction chamber 25 to prevent the fluid from bypassing the reaction chamber 25 when the side flow rate from the hydrophilic adhesive along the walls is higher than the central flow rate. For example, in an embodiment, the adhesive material or substance is not applied on, or is cut away from, the portion of the laminate close along the edges of the at least one reaction chamber 25.

With respect to Fig. 4A, once the bodily fluid sample has exited the saliva port 21 through the through hole 33, the fluid is coupled to two channels -the transfer channel 41 and the retention channel 42. A small part of the fluid travels upstream along the transfer channel 41 to a common port 43. As the common port 42 is open to air (generally 1 atm) which is a higher pressure than the channel (capillary pressure), this small part of the fluid is impeded from entering the common port 42. However, there may be a bit of capillary action along the ex-posed hydrophilic surface of the bonding layer 12 on the sides.

Reagents are later released into the common port 43 in sequence to avoid the introduction of bubbles when reagents are added. This is described later.

The rest of the fluid travels downstream along the retention channel 42 to the at least one reaction chamber channels 44 defined in the bonding layer 12 using capillary forces along the exposed side walls of the adhesive cut.

The fluid moves through the at least one reaction chambers 25 and the curve channel 44 until it reaches the capillary pump 26 and the volume of fluid/sample contained in the saliva port has been aspirated. The capillary pump 26 acts like a large waste chamber providing extra capillary pull to guarantee that the fluid flows.

The distance between the centre of the saliva port through hole 33 and the common port 43 outlet on one hand and the distance between the centre of the saliva port through hole 33 and the entrance of the first reaction chamber of the at least one reaction chambers 25 (in Fig. 4A this is the inlet of first of the four reaction chambers) is optimized experimentally to balance the hydraulic resistance and the capillary forces to ensure that the fluid travels downstream through the reaction chambers 25 rather than upstream while the reagents delivered into the common port still rapidly (< 45 s, typically) drain out of the port through the RCs to complete the assay workflow.

The width of the retention channel 42 of the at least one reaction chamber 25 is designed to be the lowest width of the channel structure in the bonding layer 12 to prevent the fluid from flowing backwards once it has entered the reaction chambers 25. This channel is consequently also the main contributor to the hydraulic resistance into the reaction chambers.

The width of the channel structure around the saliva port 21 through hole 33 is less than the diameter of the through hole 33 to expose the hydrophilic surface of the bonding layer 12 and promote flow out of the common port 23. This also assists in preventing the interruption of the workflow when the saliva hole is drained and filled with air at the atmospheric pressure.

Fig. 4B illustrates an overlap of the bonding layer 12 and the features on the bottom surface of the moulded fluidic channel structure layer 11. The feature 47 in Fig. B denotes a desirable feature for the device. The width of the outlet 47 of the at least one reaction chamber 25 or curve channel 44 (48 in Fig. 4B) in the bonding layer 12 is at least twice that of the capillary pump 26 inlet so as not to clog the channels off given the alignment tolerance for assembly. It also provides a low hydraulic resistance for the fluid exiting the bonding layer 12 channel structure. In an embodiment, the width of the outlet of the last reaction chamber 25 in the bonding layer 12 is at least 3 times larger than the pump inlet channel (0.2 -0.3 mm) to prevent misalignment from assembly tolerances and restriction for the fluid to transit from the adhesive layer to the plastic layer after incubating the reaction areas Other desirable features relate to the overlap of the bonding layer 12 and the moulded fluidic channel structure layer 11 during assembly. First, the width of the cut in the bonding layer 12 around the reaction chambers 25 is slightly higher (typically, 50 pm on each side) than the corresponding reaction chamber 25 on the defined in the moulded fluidic channel structure layer 11 (Figure 4B) to prevent the fluid from bypassing the reaction area from the stronger side flow along the walls if the adhesive is exposed to the fluid.

In the preferred embodiment, the bonding layer 12 is a double-sided pressure-activated hydrophilic tape with a total thickness of 175 pm. However, only the side in contact with the fluidic structures on the moulded part may be hydrophilic to preferably guarantee fluid propagation. The thickness defines the minimum volume of saliva to reach the pump and consider the sample loaded. An acceptable range of thickness is from 0.1 to 0.25 mm, which defines a minimum sample volume range of 6 -9 pL. The lowest possible thickness is defined by the lack of deformation (collapse) of the channel features during the pressure-driven assembly process.

A double-sided acrylic tape with a water contact angle of < 5° is suitable. Alternatively, two pieces of hydrophilic single sided tape, which has a water contact angle of < 5°, may be used joined. The two pieces of hydrophilic single sided tape can be joined together by another double-sided tape. As this double-sided tape is not in contact with the fluid, the water contact angle is not of importance. However, other types of hydrophilic tape can be used.

The fluidic channel structure layer 21 also comprises at least one reagent port 22 in its top surface. These ports facilitate the input/loading of reagent(s) into the test device In an embodiment, collapsible one-shot reagent pouches (commonly named blisters) may be used. These are made of a collapsible dome sealed at the bottom by a thin layer of metallized foil.

The at least one reagent port 22 contains features that pierce the foil at a precise point of rupture when a downwards force is exerted onto the dome by an actuator made of a moving mechanism that applies a constant compression force or, more simply, by a user's finger. This enables an entire assay to be performed without any external intervention other than the user pressing reagents in the device manually.

Fig. 5A illustrates a reagent port 22 in relation with off-the-shelf reagent pouches 51 used in the preferred embodiment. The reagent port 22 is a pocket with a diameter D, a depth h, and curvature radius r. The diameter D is equal to that of the dome of the reagent pouch. The depth h is set to 0.8 mm, in the preferred embodiment, and the curvature radius r is equal to that of the mechanical part pressing onto the dome of the reagent pouch so that there is only a small gap left between the bottom of the pocket and the actuator part at the maximum travel distance or maximum compression force. At the bottom of the reagent port, there is a reagent collection hole 52 that extends through the fluidic channel structure layer 11 to the fluidic features on the bottom side of the fluidic channel structure layer 11.

In an embodiment, located at the bottom of the reagent port 22 is at least one highaspect-ratio spike or piercing structure 53. The spikes 53 are moulded directly onto the top-most surface of the reagent port 22. While spikes are shown in the presented embodiments, in other embodiments any shape that can enable the rupturing of the reagent pouch may be used.

In the optimized implementation, the moulded spikes 53 used to rupture the foil of the reagent pouch 51 under continued compressive load and result in the release of the reagent are defined by one or more parameters, as illustrated in Fig. 5B: the maximum diameters at the base (d1) and the top (d2) of the piercing structure 53, and in particular, their ratio to define the sharpness of the feature; the height h' from the bottom to the top base that defines a gap of, typically, 0.1 mm between the top of the piercing structure 53 and the initial position of the foil at the bottom of the reagent pouch 51 set by the pocket depth h (Figure 5A); and/or the distance I between the bottom of the piercing structure 53 and the centre of the reagent collection hole 52 through which the fluid flows to the fluidic features on the other face of the moulded plastic part.

In an embodiment, the piercing feature 53 is arranged in triplicates around the collection hole 52 to mitigate against the risk of an individual spike 53 not piercing the blister pack and to increase the rate at which the reagent escapes the reagent pouch 51, while respecting the radial geometry of the fluid release mechanism.

Additionally, collection channels 54 are added to improve the efficiency of directing the fluid to the collection hole 52. They are placed at an angle to extend tangentially to the curvature of the pocket (Fig. 5B).

Under these conditions, the reagent contained in the reagent pouch 51 can be released as depicted in Fig. 5C which shows a schematic of the opening sequence and the release of the reagent fluid. When contact is made with the dome of the reagent pouch, the foil slightly deforms under the pressure of the reagent stored inside until the gap between the foil and the top of the piercing feature is bridged (step 1.). Further compression ruptures the foil when the piercing feature exerts enough tension on the foil (step. 2.). The reagent starts spilling out of the reagent pouch as the burst pressure is reached and travels downstream along the piercing feature and into the collection channels and collection hole (step 3.).

In an embodiment, the test device may couple with an actuator, or actuator indentor, configured to press the reagent pouches 51. In an embodiment, the actuator may be part of a reader device.

At the maximum travel of the actuator indentor, the gap between the indentor and the reagent pocket allows clearance for the compressed pouch (step 4.). This position is maintained until the role of the device is fulfilled in order not to create backpressure into the pouch and backflow of the reagents from the reaction areas define earlier. The indentor may have gaps at the bottom to prevent damaging the piercers even though the device is meant to be disposable, and the only risk is for a piercing feature to break too early and obstruct the transition of the fluid into the collection hole.

In the preferred embodiment, there is an array of four reagent ports 22 to accept four reagent pouches 51, such that each reagent pouch 51 can be actuated in a pre-determined sequence with set rest durations between the release of reagent at each reagent port 22.

The fluid sample moves through the collection hole 52 to a reagent channel 24 on the bottom side of the fluidic channel structure layer 11. The reagent channel 24 is coupled to the common port 23.

Fig. 6A illustrates the configuration of the at least one reagent channels 24 from the at least one reagent ports 22 to the common port 23 in a transparency model of the device. The fluid moves through the reagent channel under pressure from the burst of the pouches, or injection of the reagent. It also moves under capillary pressure, but it is the burst pressure that is the dominating cause of fluid movement.

Fig. 6B illustrates the interface between the reagent channels 24 and the common port 23.

In the presented embodiment, the reagent channels 24 are formed when the fluidic channel structure layer 11 is attached to the bonding layer 12, such that an inner surface of the reagent channels 24 is the bonding layer 12. However, the reagent channel 24 doesn't need a hydrophilic surface as it is pressure that drives the movement. However, the bonding layer 12 may comprise a hydrophilic adhesive so that further channels do not need to be cut out of the bonding layer 12, which may affect the bonding layer's 12 structural integrity.

In a reagent pouch 51, a typical volume of reagent would be 50 mL. However, not all the volume of reagent in the reagent pouch 51 will be released into the reagent port 22. Furthermore, not all the released agent will be needed for use in the test device 10. Therefore, a manner to adjust and/or control the volume of reagent delivered to the common port 23 is desirable.

In the preferred embodiment, the volumes of reagent released from the reagent pouches 51 into the reagent ports 22 were measured experimentally and found to be a minimum of 28 pL at each of the four reagent ports 22.

The reagent channel(s) 24 from the reagent port(s) 22 to the common port 23 have a static role to create a waste or unused volume of reagent that is not required for the assay. The measured released volume serves as a basis for adjusting the dimensions of the reagent channels from each reagent port where the reagent is released, for example by the piercing structures 53 (Fig. 5A-5B), to the common port 23.

For this reason, the reagent channels 24 are separated by at least 1.5 mm from each other to reduce leakage paths (Fig. 6A) and have a much smaller (typically 1-2%) cross-sectional area than the common port 23 wall with which they intersect (Figure 6B) so that the capillary pressure differential for causing the fluid to flow from the common port 23 to the reagent channel 24 is very high.

For a given cross-sectional area, the total wasted volume for each reagent channel 24 is defined by its length (Fig. 6A). The additional volume in the collection hole 52 (Figure 5B) is a common dead volume of typically 1.0 pL.

In addition, the bonding layer 12 underneath the channels and, in particular, at the interface with the common port 23 is structured (Fig. 7B) so as to prevent fluid from moving from the common port 23 to the reagent channel 24. This sis discussed further below.

Fig. 7A illustrates the structure of the common port 23. The common port 23 comprises a central chamber 71, an outlet 72 and at least one angled inlet 73. While Fig. 7A shows 4 inlets, the common port 23 can comprise of one or more inlets 73, depending on the number of reagents to be added, and the number of reagent ports 22. For example, for four reagents to be added there would be four reagent ports 22, four reagent channels 24 and four inlets 73.

The burst pressure to rupture the reagent pouches 51 causes flow rates in the order of e.g., 1000-2000 pL/min. However, the capillary drive-section of the device (from the outlet of the common port to the capillary pump) may only tolerate much lower flow rates (e.g. 1000-2000 pL/min).

The role of the common port 23 is twofold. Firstly, it absorbs each reagent injected into the reagent port 22 with a high velocity and high pressure (flow rates: e.g., 1000-2000 pl./min) due to the high force applied to cause the rupture of the reagent pouch 51, or, alternatively, a high force applied to inject the fluid using a syringe or other means.

Secondly, the common port 23 initiates the slower release (typical flow rates: 50-100 pL/min) of the reagent to the capillary-driven section of the device for the sequence of interactions in the reaction areas desired to complete the assay.

In this way the common port adapts the high-pressure sections of the device (reagent ports and reagent channels) to the capillary driven sections of the reagent chambers 25 and/or capillary pump 26.

The common port 23 functions as a dynamic reservoir using one or more of the below: angled inlets 73 that direct the fluid to the outlet 72. This guarantees that the fluid (reagent) injected at the reagent ports 22, for example from the reagent pouches 51, is directed to the outlet 72 (at the bottom end of the common port) and that the reagent starts draining from the common port 23 rapidly (i.e., typically within 40 s) so as to be quickly delivered to the reaction chambers 25 downstream without affecting the desired incubation durations. In the embodiment of Fig. 7A, the first 3 reagents are delivered at increasing angles on the left edge whereas the final reagent is delivered from the top edge. In an embodiment, the inlet on the top edge is used to receive the last reagent added (detection substrate). In alternate embodiments, the inlets 73 may be arranged on the same side (ie. left edge). The placement of the inlets 73 is preferably such that they are separated enough to leave enough space for proper sealing without leaks between channels.

an asymmetric geometry where an inner wall of the common port 23 is further away from the centerline (a line cutting through the outlet) so that the velocity of the reagents injected at high pressures can be reduced rapidly without causing excessive turbulent flow to fill the port at a much slower rate and drain it. The bounce back from a wall opposite the inlet allows for kinetic energy to be absorbed before entering the outlet of the common port. This may prevent flooding of the device; and/or a porous hydrophobic membrane for venting. It covers the top of the common port 23 (opening for common port 23 in the fluidic channel structure layer 11) and allows air to escape when the fluid fills the common port 23 without letting the fluid through due to a restricted pore size (e.g., 20 -100 pm diameter). An alternative is to use a vent channel and vent hole, but this presents two risks: i) the channel can fill up with fluid and no longer be able to vent the port and ii) reagents can squirt out and present a potential health and safety risk for the user. The rupturing of the reagent pouches will cause bubbles to form in the reagent. These bubbles will have a negative effect on the assay performed by the test device if the bubbles are allowed to move down into the reaction chambers 25. The membrane of the common port allows potential bubbles formed during reagent injection to burst. The membrane may cover the entire top surface area of the common port or a portion thereof.

The membrane may be a membrane suitable for use in cell culture chambers that allow CO2 to escape but prevent the cell culture from leaving the chamber.

In an embodiment, the membrane is hydrophobic so that liquid doesn't wet the membrane. The membrane may be formed of rayon, which is a type of Teflon®, which is hydrophobic.

Then, once the reagent has been added and draining has started, the common port 23 acts as a passive reservoir with a controlled capacity (slightly higher than the maximum released volume from each reagent pouch 51). The capacity of the common port 23 is preferably in the order of 34 pL. For example, the fluidic channel structure layer 11 may define a capacity of 31 pL and the additional thickness of the bonding layer11 provides a further 3 pL. This can be achieved by using: a tapered narrowed outlet 72 to channel the fluid delivered at high pressures from the reagent pouches 51into the outlet 72 (see Figure 7B); and/or the transfer channel 41 defined in the bonding layer 12 with a width between 0.2 and 0.4 mm for a height around 0.1 -0.2 mm. The cross-sectional area is the smallest in the moulded fluidic channel structure layer 11 so that the capillary pressure is highest and ensures that bubbles formed in the common port 23 do not enter the capillary-driven section of the device downstream.

Fig. 7B illustrates use of the bonding layer 12 to structure the connection between the reagent channels 24 and the common port 23 to prevent the spilling of reagents from one reagent channel 24 to the next.

Fig. 7B also illustrates an alternate concept of recessing the bonding layer 12 on the right edge to further promote movement into the outlet 72. The adhesive cut around the feature defining the common port 23 in the fluidic channel structure layer 11 can also be slightly recessed on the right edge to prevent the fluid from travelling along the right edge of the common port 23 and promote movement into the outlet channel (Fig. 7B).

Fig. 8 illustrates the different steps of the injection of reagent in the common port 23 via the reagent channels and the subsequent draining into the transfer channel 41 defined in the bonding layer 12. One or more steps may be implemented in an embodiment: Step 1: the fluid enters the common port 23 at a high front velocity (high pressure) through inlets on an inlet wall on the common port; Step 2: the fluid hits the opposing wall that extends further away from the centerline (from the outlet, center of arc defining the bottom edge of the port) of the common port 23 (asymmetry) than the inlet wall and at a lower angle than the inlet wall to rapidly reduce the front velocity and redirect the fluid to fill the common port 23 with a more even fluid front by redistributing the jet of fluid hitting it from the reagent channel to several directions; Step 3: the common port 23 fills up while the fluid does not spill into the next channel 25. This is achieved by removing the more hydrophilic surface of the bonding layer 21 at the bottom of the channels 24 at the inlets of common port 23 (cut line interface shown in red in Fig 8); Step 4: draining is initiated when the hydrostatic pressure of the fluid in the port overcomes the capillary pressure into the transfer channel 41; Step 5: draining is complete, and the common port 23 is emptied. The fluid in the reagent channel 24 (waste volume) is not drained because of the pressure is in the reagent channel 24 is lower than in the common port (which is open to air, so around 1 atm); and/or Step 6: the next reagent in the assay sequence is added.

The path to the reaction chambers is mostly defined in the bonding layer 12 between the fluidic channel structure layer 11 and the electrode layer 13 (Fig. 1). The reaction chambers 25 are defined in the fluidic channel structure layer 11.

Fig. 9 illustrates the fluidic structures in the bonding layer 12 allowing reagents to move from the common port 23 to the reaction chambers 25. A narrower retention channel 42 is added to ensure the fluid does not travel upstream back into the common port 23 by capillarity, i.e. the retention channel 42 is narrower than the curve channel 44 and the transfer channel 41. This adjusts the capillary pressure in the retention channel 42 so that it is lower than the pressure in the common port 23 and may thus guarantee in embodiments that the fluid does not flow back to the common port 23.

In the preferred embodiment, the fluidic channel structure layer 11 only contains four preferably recessed chambers 25 with a depth of e.g. 0.2 -0.5 mm but the number of chambers is only limited by the size of the final device (Fig. 9). The chambers 25 need not be identical. In a preferred embodiment, the chambers are identical. This is preferred if measuring repeats or related quantities so that the flow conditions on the funcfionalized (antibody-coated) surface are the same. Each reaction chamber 25 is separated by a precise distance that would allow simultaneous deposition of reagents using different capillaries or nozzles in commercial dispensing systems. The distance between reaction chambers 25 may be adjusted to be compatible with a system for dispensing. Separating the chambers by the same amount ensures that the reagents could be dispensed in each chamber simultaneously and speed-up manufacturing.

In the preferred embodiment, a first reaction chamber 25 contains a negative control, a second reaction chamber 25 contains a positive control while the third and fourth reaction chambers 25 provide repeats of the sample to be measured. Alternatively, the second reaction chamber 25 and the third reaction chamber may be configured to allow measurement of different reagents. Furthermore, the order of the chambers may be different to that described. The dimensions of the chamber are tailored to the size of the sensor electrodes and the desired electrode areas to obtain an acceptable signal-to-noise ratio (see further details on electrodes below). The recessed reaction chambers 25 provide a controlled deposition area of the reagents during the derivafizafion of the surface with assay-specific reagents and prevent the spilling of these reagents in the device.

The flow of reagent and saliva to the reaction chambers 25 is via the transfer channel 41, the retention channel 42 and the curve channel 44.

As mentioned previously, the width of the transfer channel 41 around the through hole 33 of the saliva port 21 is slightly lower than the through hole 33 diameter to ensure the presence of a strong side flow from the exposed hydrophilic adhesive that guarantees that the flow is not interrupted by the presence of a bubble inside the saliva hole.

As mentioned previously, the width of the retention channel 42 upstream of the first reaction chamber 25 is designed to be the lowest width of the channel structure in the bonding layer 12 to prevent the fluid from flowing backwards once it has entered the reaction chambers. This channel is consequently also the main contributor to the hydraulic resistance into the reaction chambers 25.

In the curve channel 44, the turns between the reaction chambers 25 provide a higher hydraulic resistance to limit the contamination between samples measured in each chamber. The adhesive is recessed along both edges of each reaction chamber 25 to prevent the fluid from bypassing the reaction chamber when the side flow rate from the hydrophilic adhesive along the walls is higher than the central flow rate.

The reaction chambers are coupled to the capillary pump Fig 10A shows a footprint of the capillary pump and Fig. 10B illustrates details of the diamond pillars at the inlet of the pump.

Preferably, the capillary pump 26 is made of several thousands of preferably diamond-shaped pillars 101 separated by e. g. about 0.15-0.20 mm to from a network of capillary channels 102 for the fluid waste from the reaction chambers 25 to fill up progressively as each reagent is added to the device 10. The structure of the array of pillars 101 in an embodiment may be identical to that described in our earlier application: Microfluidic POC Assay, GB 2003979.8. The array is extended along the length (major dimension) of the pump. The depth is adjusted within e. g. 0.28 -0.35 mm to tune the capacity of the pump to the volume of fluid dispensed out of the reagent pouches and added form the saliva port considering the volume of the adjustment channels and other fluidic features in the device. The typical capacities are between e. g. 150 and 190 pL. In the preferred embodiment, the capacity is around 160 pL so that there is approximately 30 pL of unused volume in the pump at the completion of the assay.

Preferably, the capillary pump 26 has a capillary pressure greater than the rest of micro-fluidic circuit of test device 26. The capillary pump 26 may comprise a vent coupled to the outside environment.

The hydrophilic properties of the bonding layer 12 are utilised by the capillary pump 26 to promote the flow of fluid into the capillary pump 26.

Figs. 11A -11D illustrate the electrode (sensor) layer 13 and the types of sensors used.

The electrode layer 13 is comprised of a plurality of sensors, which when the device 10 is inserted into a reader device, make electrical contact with the reader device to facilitate measurements. The sensors may comprise one or more of: * a strip detection sensor 110 comprising a current loop that when closed upon proper insertion of the test device into a reader, closes a circuit that indicates that the sequence outlined e. g. in Fig. 12 should be commenced. The strip detection sensor 110 (Fig. 11B) is engaged when a current loop between two selected tracks is closed in the reader device upon insertion of the test strip and the connection of two selected connector pins by a low resistance (typically 20-250 0). The current loop may trigger an indicator on the reader device to indicate to the user to begin the sequence; * a common port sensor 111 comprising two electrodes to detect the presence or absence of fluid in the common port 23. The electrodes are connected near the outlet of the common port (Fig. 11C). This is used as a failure detection mechanism should the reagent not have been properly released from the reagent pouches. When no fluid is present in the common port, there is a very low leakage current detected between these electrodes (using either chronoamperometry or by monitoring the open circuit voltage in a 2-electrode configuration). On the contrary, a much higher current is detected when fluid bridges both electrodes if a sufficient volume of regent has entered the common port. Alternatively, the electrodes can be used to measure the impedance in the cell constituted by the common port. Similarly, the successful delivery of fluid is determined by a significant change in the current flowing between both electrodes rising above the noise background level; and/or * reaction chamber sensors 112 to make a measurement in a reaction chamber 25. Each reaction chamber sensor comprises a working (WE) electrode, a reference (RE) electrode which occupy typically 60% of the overall length of each reaction chamber. There is one set of electrodes for each reaction chamber. Each reaction chamber sensor further comprises a counter electrode (CE). The reaction chamber sensors may share a common counter electrode (CE). In an embodiment, a standard 3-electrode electrochemical cell is formed using a common counter electrode (CE) that occupies another 20% of the overall length of each reaction chamber. The area of the working electrode is optimized to obtain a large enough electrode area to obtain a signal-to-noise ratio sufficient for the detection of low analyte levels. An alternative embodiment may use a common RE electrode, which is shared across the different reaction chambers, to simplify the electric connectivity.

In the preferred embodiment, the electrode layer 13 is made by laser ablating a polyethylene (PET) sheet sputtered with a thin gold film using a fiber laser, but other manufacturing methods including stereolithography or screen-printing can be used.

Advantageously, an embodiment of the device 10 requires minimum intervention from the user aside from inserting the strip in the reader/actuator system and adding the sample (e.g., saliva) to the saliva port 21 using either a pipette, a droplet dispensing tool, or a specific saliva collection tool (e.g., see GB 2104622.2), for example according to steps shown in Fig. 12.

Fig. 12 illustrates a simplified flow diagram of the order of steps, one or more of which may be required to use the preferred embodiment. The user may only be required to perform the first two operations.

Embodiments of the device generally use first the burst pressure from the reagents encapsulated inside collapsible pouches, then capillary forces to propagate the fluid. Consequently, an embodiment can be used without any external apparatus if the user presses each reagent pouch manually in a controlled sequence. The use of the actuator/reader device may ensure a more precise and/or automated use of the device to provide a result with enhanced accuracy.

At S121, the user inserts the device 10 into a reader device. At S122, the user adds a sample to the saliva port. The reader device then waits e.g., between 5 to 20 minutes before adding a first reagent at S123. The reader device may comprise an actuator configured to press the reagent pouches 51 (blisters). The reader device waits another e.g., 5 to 20 minutes before adding a first wash reagent at S124. Then, after waiting another e.g., 1-10 minutes, the reader device adds a second wash reagent and waits for e.g,. 110 minutes. At S126, the reader device adds an electrochemical substrate, and after e.g., 5 -20 minutes at S127 the reader device measures each chamber in sequence. At S128, the reader device returns the analyte concentration.

In an embodiment, the first reagent is a hormone conjugate that fills up the free sites on the coated antibody not already taken by the actual hormone from the sample. This conjugate is linked to an enzyme that later oxidizes the substrate (TMB) and the complementary signal is measured. The first wash regent is a rinse buffer meant to remove nonspecifically bound conjugate molecules as well as hormones (and other impurities) from the sample.

In general, both wash the first wash reagent, and the second wash reagent are the same but not necessarily.

Fig. 13 illustrates a reader device 1300 for use with the test device 100 according to an embodiment. The reader device 1300 may be used with the test device 100 to carry out the steps of Fig. 12. The reader device 1300 comprises a reader port 1303 into which the test device 100 is inserted. The reader device 1300 also comprises a measurement unit 1302 to interface with the strip detection sensor 110, the common port sensor 111 and the reaction chamber sensors 112 to facilitate measurements. In an embodiment, the reader device 1300 comprises an actuator 1301 which can be used to couple to the reagent pouches 51 and press the pouches 51 so that they burst/rupture.

In a preferred embodiment, the saliva port cavity contains a protruding structure that pierces a sample collection tool (e.g., for saliva, e.g. GB 2104622.2). The sloped bottom of the port remains to promote the wetting of the through hole transferring the fluid to the fluidic circuit on the other face of the device. Alternatively, the saliva port does not contain a protrusion and would allow for dispensed addition of saliva using either a dropper or a micropipette or another apparatus capable to dispensing a volume of between 10 and 40 microlitres.

Due to the use of capillarity to fill the device with sample and reagents as well as the possibility to integrated regent capsules (or blisters) available commercially (off-the-shelf or customized to desired dimensions and content), an embodiment can perform a biochemical assay on bodily fluid (e.g., saliva, urine, serum) with minimal help from external hardware and very limited user input. This is a clear improvement of embodiments over existing methods to perform such assay using microtiter plates or commercial tubes.

In the preferred embodiment, the integrated test strip operates in several major steps such as one or more of the example steps a) -e) and features thereof below (preferably in the order laid out below): a) Loading with bodily fluid sample, e.g. saliva: * The profiled saliva port 21 can contain e.g., 10-40 pL of fluid while maximizing height of hydrostatic fluid column; * The saliva port 21 has a short (e.g., 0.3-1.0 mm) through-hole 33 to connect to fluidic circuitry on the bottom surface of the fluidic channel structure layer 11; * The saliva port has an angled bottom conical section 32 to favour wetting of the through hole 33 even for hydrophobic materials (e.g., polystyrene); and/or * Blocking with hydrophilic coating to favour propagation of fluid into through hole.

b) Transfer to reaction chambers 25: * Fluidic channels (transfer channel 41, retention channel 42 and curve channel 44 defined in pressure-sensitive bonding layer 12 (thickness e.g., 0.15 -0.2 mm); * Balances hydraulic resistance upstream (to common port 23) and downstream (to reaction chambers 25) to favour downstream flow; and/or * Fluid moved by capillarity on hydrophilic adhesive side walls before reaching capillary pump 26.

c) Capillary pump 26 machined directly into the device: * Composed of multiple diamond-like pillars 101; * Creates network of channels 102 to be filled by capillarity; * Vented by connection to the outside world; and/or * Capacity controlled by area and depth, can be up to e.g., 150 pL.

d) Reagent pouch 51 (blister) piercing interface: * Manufacturable feature to pierce sealing foil of reagent pouches 51; * Three piercing structure 53 to mitigate failure; * Recessed into top surface of the fluidic channel structure layer 11 to protect from unwanted contact; and/or * Secondary collection channels 54 that interface with collection hole 52 to fluidic circuitry on bottom surface of the fluidic channel structure layer 11.

e) Reagent channels 24 and common port 23: * Absorb high pressure from bursting of reagent pouches 51; * Reagent channel 24 height can be adjust to adjust the volume of reagent needed; * Angled interface between reagent channels and inlets 73 of the common port 23 to favour rapid (e.g <30 s) draining of the common port content into reaction chambers; * Asymmetric common port 23 shape to favour rapid drain into outlet 72 on one edge and prevent turbulent flow on opposite edge to reduce bubble formation; * Vented common port 23 (e.g., using a hydrophobic porous membrane) to absorb burst pressure and confine reagent to outlet 72; and/or * Narrow transfer channel 41 defined in bonding layer 12 to retain large bubbles and prevent backflow from reaction chambers 25.

f) Multiple identical reaction chambers 25: * Allow the integration of positive and negative controls as well as sample duplicates; * Configured for compatibility with commercial dispensing systems; * Connected by channels defined in bonding layer with controlled hydraulic resistance to favour fast loading of sample (e.g. <90s); and/or * Recessed deposition cavity in device to aid surface functionalizafion for biochemical assay and maintain a set distance from sensor regardless of adhesive layer compression during assembly.

A preferred embodiment may provide any one or more of the following advantages: - the coupling between the capillary-driven side of the device where the assay steps take place and the pressure-driven side where reagents are stored and released. This may be achieved by balancing the hydraulic resistance from the sample addition port so that the sample travels slightly upstream to contact the common port but then progresses downstream to reach the capillary pump; - the architecture of the common port including the asymmetry of the main cham-ber (reservoir) and the angle of the input channels relative to the outlet channel to create a dynamic of filling and draining each reagent from the common port; - a vented common port chamber that absorbs the pressure from the reagent blister reservoirs and rapidly (e.g. < 30 s) distributes the reagent volume into the capillary sub-system; - the use of a porous membrane to vent the common port. This is an improvement over the prior art with a channel and a vent hole and allows for venting across the whole area of the common port and the minimization of bubbles; - a transfer channel structure defined in the bonding layer to couple the common port to the capillary-driven reaction chambers of the device; - a geometry for the sample port that allows, preferably guarantees, a fast (e.g. <90 s) loading into the device by capillarity; and/or - the multiplexing into reaction areas that are recessed into the device thereby maintaining a set distance and geometry between the hard plastic device and the sensor during the assembly steps.

The preferred embodiment may include features of interest such as: - the integration of existing commercial blister capsules (or reservoir) pre-filled with reagents and sealed; - the use of an adhesive layer that bonds the device to the measurement layer (e.g., electrodes) and seals it to the outside world but also contains features desired for fluid management; - a reaction chamber that assists the manufacturing of the device (deposition aid for surface modification with targets) and reduces the noise in the measurement; and/or - a design amenable to injection molding as well as high-resolution 3D printing.

Fig.14A shows a top view of the microfluidic test device with the reagent pouches according to an embodiment.

Fig.14B shows a bottom view of the microfluidic test device with the reagent pouches according to an embodiment.

Fig. 15A shows an overlay of the moulded fluidic channel structure layer and the bonding layer according to an embodiment.

Fig. 158 shows cross sections corresponding to Fig. 15A.

An advantage of the device is to provide an automated reagent dispensing method, which constitutes a desirable alternative to pipetfing each reagent manually into the device. Pipetting each reagent manually into the device requires trained personnel and is prone to user error. In the current implementation, the user is only required to deposit an appropriate volume of sample such as, but not limited to, saliva in the sample port using a pipette or a droplet dispensing tool. The device can also be coupled to a saliva collection tool that further automates the assay.

The present disclosure describes the operation of the device by a reader/actuator to reduce user intervention. However, the use of embedded collapsible pouches and capillary-driven flow makes it possible to use the device manually by pushing each reagent pouch open in sequence. The readout requires an electrochemical readout, but in an embodiment the device could operate as a colorimetric assay where the result is simply read using the camera present in most smartphones.

Alternative implementation: The preferred embodiment uses a sensor layer made by tailoring a thin gold film into appropriate tracks. However, the readout could be colorimetric or fluorometric. This would require replacing the sensor layer by an optically transparent window and making the plastic fluidic device out of a low-fluorescence polycarbonate material or cyclic olefin copolymer (COC). These are also standard materials available for injection molding and mass production. In this alternative, the difference relies on the use of an analyte with a shift in its absorption spectrum in the UV-visible domain (for colorimetric applications) or in its fluorescence emission spectrum when excited at a set wavelength (for fluorescence detection). For colorimetric detection, the reader device would be comprised of an excitation LED source (either one per reaction chamber or coupled to each chamber using fiberoptics in the form of a multi-furcated cable or a bundle) and a detector array (such as an avalanche photodiode or CCD array) sandwiching the optically transparent device that acts as an absorption cell. The assay result is obtained by monitoring the change in absorbance at a set wavelength (given the LED emission spectrum and emission filter). For fluorometric detection, the reader device would use a collimated high intensity light source (e.g., a laser diode or small compact diode-pumped solid-state laser (DPSSL) and similar detector (CCD or CMOS array, photomulfiplier tube, or compact spectrometer). If low sensitivity is not required, the detection could be done using a colorimetric analyte and a high-resolution image of the diverse rection chambers obtained by a smartphone. This would be particularly advantageous in low-resource settings or for handled screening applications.

The preferred embodiment of the present invention is to carry out the workflow of an ELISA-based assay for hormone detection in saliva samples. However, the configuration chosen using collapsible reagent pouches and a common port can also be used to mix reagents in the common port and drain them by further mixing with the content of the saliva port in a laminar flow from the common port to the capillary pump, where they can be retrieved for further reaction steps or analysis. This implies the presented device can also be used for sample preparation in diverse assays. This would, among other things, require the sequence of actuation of each reagent pouches to be adapted for the diagram presented in Fig 12.

Notes on hydraulic resistance balancina Both upstream and downstream sections may be overly simplified as two channels with rectangular cross-sections with hydraulic resistances Rhupstream and Rhdownstream, respectively.

For water, rj=1 mPas: Dupstream _ 12(L=3mm) 1 = 2.85 1011 Pa. s. m-3 At h 1-0.63(h=0.113mm/w=0.33mm) (h=0.18mm) 3 (w=0.33mm) pp downstream = 12n(L=27mm) 1 -8.73 1011 Pa. s. m-3 * i h 1-0.63(h=0.113mm/w=0.75mm) (h=0.18mm) 3 (w=0.75mm) Equation from Source: Bruus H., Theoretical microfluidics, Lecture Notes, TU Denmark, 3' Ed., 2006: Based on just that, the resistance downstream is higher.

However, several things 1) The capillary force along the longer channels downstream (curve channel, capillary pump etc). Simple formulas are only available to get the capillary pressure at the point of change in the cross-section as: ( cos Source: Zimmermann M. eta!, Lab Chip (2007), 7: 119 There is no easy way to calculate the capillary force sensed over the entire channel length but the downstream section being much longer, the capillary force is much higher (as judged by the hydrophilic surface area (side walls) in contact with the fluid along the channel).

2) Every turn in the downstream channel (neglected in the equation below) add hydraulic resistance. In addition, the capillary pump has its own complex hydraulic resistance given by: with m=n(2n-1)/ch, ch=channel height, and cw=channel width Source: Vasilakis N. et a!, Microfluid Nanofluid (2017), 21:103 When limited to the 3rd order, a graphical method gives approximately 8.5 1015 Pa.s.m-3.

3) An advantageous difference in embodiments is that the common port (upstream) is open to air with P=1 atm whereas the downstream section is connected to the capillary pump where a high negative pressure is available due do the capillary action inside the pump.

All these reasons are why the fluid stops upstream at the common port but continues all the way to the capillary pump downstream until the content of the saliva port is emptied. The complexity of the situation is why this could not be easily modelled and had to be adjusted experimentally using trial and error prototyping.

Any measurements indicated throughout the description or in the figures are for only example purposes, and other dimensions may be used

Claims

CLAIMS: 1. A test device (10) for bodily fluids comprising: at least one reagent port (22) configured to accept at least one reagent under pressure from a reagent pouch (51); a sample port (21) configured to accept a bodily fluid sample; a common port (23), comprising an output (72) and at least one input (73), each at least one input (73) of the common port (23) coupled to one reagent port (22) of the at least one reagent port (22) by a reagent channel (24), the output of the common port (23) coupled to the sample port (21) by a transfer channel (41); and at least one reaction chamber (25) comprising a plurality of electrodes to perform at least part of a biosensing test of a substrate solution, the substrate solution comprising the bodily fluid sample and the at least one reagent, the reaction chamber functionalized with one or more bioreceptors for binding to a target analyte, each reaction chamber of the at least one reaction chamber (25) coupled to the sample port (21) by a retention channel (42), wherein a top surface of the common port (23) comprises a gas-permeable membrane configured to allow gas to pass through but prevent fluid.
2. The microfluidic test device (10) of claim 1 wherein the at least one input (73) of the common port (23) is angled in a direction towards the outlet (72) of the common port to facilitate rapid draining of the common port (23).
3. The microfluidic test device (10) of any preceding claim wherein the common port (23) has an asymmetric geometry about an axis passing through the centre of the outlet (72) of the common port (23), wherein the common port (23) comprises an opposing wall opposite an inlet wall comprising at least one inlet of the common port (23), the inlet coupled to the reagent channel (24), wherein the opposing wall extends from the outlet at a lower angle that the inlet wall.
4. The microfluidic test device (10) of any preceding claim wherein the common port (23) has a capacity greater than a maximum released volume from each reagent pouch (51).
5. The microfluidic test device (10) of any preceding claim wherein a capacity of the at least one reagent channel (24) is configured to be equal to a volume of unneeded reagent from the reagent pouch (51).
6. The microfluidic test device (10) of any preceding claim wherein an inner surface of the reagent channel (24) comprises a hydrophilic material, wherein inner surfaces of the common port (23) do not comprise a hydrophilic material.
7. The microfluidic test device (10) of any preceding claim wherein a cross-sectional area of the retention channel is smaller than a cross-sectional area of the inlet of the at least one reaction chamber (25).
8. The microfluidic test device (10) of any preceding claim wherein a cross sectional area of the transfer channel (41) is smaller than a cross sectional area of the at least one reagent channel (24) and a cross sectional area of the inlet of chamber of the common port.
9. The microfluidic test device (10) of any preceding claim wherein the reagent port further comprises at least one piercing structure configured to burst the reagent pouch when the reagent pouch is coupled with the reagent port and an external pressure is applied to the reagent pouch.
10. The microfluidic test device (10) of any preceding claim wherein the sample port comprises a conical-like section (22) and a through-hole (33), wherein the conical-like section (22) comprises a surface that forms an angle of at least 65 ° with an axis passing through the centre of the through-hole (33) in the direction of fluid flow.
11. The microfluidic test device of claim 10 wherein the sample port further comprises a cylindrical-like part (31), the conical-like section (22) located between the cylindrical-like part (31) and the through-hole (33).
12. The microfluidic test device (10) of any preceding claim wherein an inner surface of the sample port comprises a hydrophilic material.
13. The microfluidic test device (10) of any preceding claim wherein the at least one reaction chamber (25) comprises at least two reaction chambers, the reaction chambers coupled together by a curve channel, wherein the curve channel comprises at least one turn.
14. The microfluidic test device of claim 13 wherein the at least one reaction chamber (25) comprises at least four reaction chambers.
15. The microfluidic test device (10) of any preceding claim wherein a capillary pressure differential from the reagent channel (24) to the common port (23) prevents flow from the common port (23) to the reagent channel (24).
16. The microfluidic test device (10) of any preceding claim wherein the at least one reagent channels (24) are separated by a distance such that there are no leakage paths between the reagent channels (24).
17. The microfluidic test device (10) of any preceding claim further comprising a capillary pump (26), the capillary pump coupled to the at least one reaction chamber.
18. The microfluidic test device (10) of any preceding claim, wherein the sample port contains a protruding structure configured to pierce a sample collection tool.
19. A fluid sample test system comprising the microfluidic test device (10) of any preceding claim and a reader device for controlling at least one of the test electrodes to perform the at least part of the biosensing test, and to output a result of the biosensing test.
20. Use of the microfluidic test device of any one of claims 1 to 18 or the test system of claim 19, to perform an ELISA or ELONA test.
21. The use according to claim 20, comprising: (i) receiving a bodily fluid sample in the sample port; and (ii) receiving a reagent in the at least one reagent port.