Patent Application
20210229090
The invention relates to a point-of-care cartridge. In particular the invention relates to a point-of-care diagnostic assay system based on centrifugal microfluidic technology.
Manual processing to determine the biochemical content of various types of samples, is cost-prohibitive in many applications and is also prone to errors. Automation is also cost-prohibitive in many applications, and is inappropriate as currently practiced—using, for example, liquid handling robots—for applications such as point-of-care or doctor's office analysis. As a result, there is an unmet is need to provide sample processing for biochemical assays that is less expensive and less prone to error than current automation or manual processing.
Typically it is very difficult to move fluids radially inward using centrifugal microfluidics as the primary means of fluid movement. This can limit/restrict the options available to allow a sequential assay to be performed.
Certain point-of-care diagnostic assay systems based on centrifugal microfluidic technology are quite good at performing the necessary integrated sample preparation and assay measurement steps. Such a centrifugal microfluidic platform with optical detection allows for a variety of assay technologies to be implemented in parallel using a single instrument and disposable cartridges Examples of point-of-care diagnostic assay systems include U.S. Pat. No. 9,182,384B2 (Roche), U.S. Pat. No. 8,415,140B2 (Panasonic), U.S. Pat. No. 8,846,380 (Infopia), U.S. Pat. No. 5,591,643 (Abaxis), U.S. Pat. No. 5,409,665 (Abaxis).
US Patent Publication No. US 2010/074801 describes an analyser comprising a microchip coupled to a motor, where the microchip acquires a liquid sample by means of capillary action. The microchip overcomes the limitation of using capillary action to move a liquid sample by providing a structure which reduces capillary pressure. This is achieved by providing each channel with an adjoining cavity open to atmospheric pressure, which acts so as to prevent an increase in capillary pressure as the fluid length increases. Thus, in one embodiment of the invention, the microchip structure comprises an inlet for collecting a liquid sample, a capillary cavity for holding a predetermined amount of the liquid sample, a single holding chamber having an analytical reagent, a measuring chamber for measuring the mixture of the liquid sample and the reagent, a channel communicating with the holding chamber and the measuring chamber, and a channel connecting the measuring chamber with an atmospheric vent. In use, a liquid sample in the capillary cavity is transferred by centrifugal force into the holding chamber, where it is mixed with the analytical reagent. This mixture is then transferred out of the holding chamber to the inlet of the measuring chamber by capillary force, from where it is transferred into the measuring chamber itself by rotation of the analyser. At the measuring chamber, the concentration of a component of the liquid sample is measured. Accordingly, it will be understood that in this patent document, the microchip structure is configured such that once the holding chamber has delivered the mixture of the single reagent and the liquid sample to the measuring chamber, the mixture cannot be returned to the holding chamber.
US Patent Publication No. US 2015/104814 discloses a sample analysis apparatus for whole blood separation. It comprises a rotatable microfluidic apparatus which comprises a sample chamber for accommodating a sample, a channel that provides a path through which the sample flows, and a valve for opening the channel, which is coupled to a valve driver and a control unit. A separation chamber receives a sample flowing from the sample chamber due to centrifugal force, while a collection chamber for collecting target cells is connected to the separation chamber. In use, the apparatus is rotated to separate the sample into a plurality of layers in the separation chamber according to density gradients of materials in the sample, such as for example a DGM layer, an RBC layer, a WBC layer and a plasma layer. The target material located in the lowermost portion of the separation chamber along with the DGM is then transported to the collection chamber for recovery.
International Patent Publication No. WO 2009/016811 describes a device for analysing a liquid collected from an organism. The device comprises a plurality of individual cuvettes, wherein each cuvette measures a different phase of a reaction. US Patent Publication No. US2017138972 simply describes the use of gravity and centrifugal forces to transfer fluids between three reaction chambers in order to provide multiple washing steps to separate a composite from unbound or unreacted substances.
It is therefore an object to provide an improved point-of-care diagnostic assay systems based on centrifugal microfluidic technology.
According to the invention, there is provided, as set out in the appended claims, a microfluidic system comprising:
In one embodiment the first zone is positioned at a radial extent and at a furthest point from the centre of rotation of the reaction chamber.
In one embodiment the second zone is positioned radially inward with respect to the first zone and comprises first reagent spot location R1.
In one embodiment wherein the third zone is positioned between the most radially inward end of the reaction chamber and the radial inward position of the second zone and the third zone comprises a second reagent spot location R2.
In one embodiment there is provided a first separate rehydration chamber to rehydrate an R1 reagent (R1-X) or a different reagent.
In one embodiment there is provided a buffer metering chamber coupled to the first separate rehydration chamber configured to meter a pre-defined volume of buffer solution for transfer to the first separate rehydration chamber to rehydrate the R1 reagent (R1-X) or a different reagent in the rehydration chamber.
In one embodiment there is provided a second separate rehydration chamber to rehydrate an R2 reagent (R2-Y) or a different reagent.
In one embodiment there is provided two or more reaction chambers.
In one embodiment the reaction chamber comprises at least one of an oblong shape; a circular shape, a square shape; a zigzag shape or a cross shape.
In one embodiment the first zone comprises a cuvette and is positioned adjacent to the outer diameter of the cartridge.
It will be appreciated the cartridge of the invention provides a number of advantages over the prior art:
In one embodiment the first detection zone comprises a cuvette and positioned at the radial extent of the V shaped reaction chamber.
In one embodiment the V shaped chamber extends radially inward on two sides to create two zones that can be independently filled with fluid to define the second zone and third zone.
In one embodiment the second and/or third zone comprises a reagent storage and/or rehydration zones.
In one embodiment the second and/or third zone comprises a region adapted to be optically interrogated.
In one embodiment the cartridge is positioned and configured to rotate at a velocity such that a combination of centrifugal force and gravity moves the fluid sample radially outward and inward respectively.
In one embodiment the cartridge rotates at a velocity such that the relative centrifugal force (RCF) is greater than gravity, and the fluid sample can be moved radially outward on the cartridge.
In one embodiment the centrifugal force ensures that no fluid reaches the second zone or third zone.
In one embodiment the cartridge is stationary or rotating slowly, gravity will influence the fluid and move the fluid towards the second zone or third zone.
In one embodiment the cartridge is rotated or agitated on an inclined plane with respect to a horizontal plane to create a downward slope for the fluid sample to flow under the influence of gravity.
In one embodiment, the cartridge is further configurable to be agitated to overcome any effects of surface tension that may prevent the fluid from flowing under the influence of gravity.
In one embodiment the cartridge rotates on an inclined plane at an angle of θi from the horizontal plane and wherein the angle is between 10° to 60°.
In one embodiment a buffer reservoir is positioned close to the centre of rotation of the cartridge and a module configured for applying a sample directly to the cartridge.
In one embodiment the dominant force on the fluid sample meniscus is the centrifugal force such that the centrifugal force is parallel to the upper and lower surface of the first detection zone to provide a meniscus evenly on both surfaces.
In one embodiment the second zone comprises a dried reagent.
In one embodiment the third zone comprises a dried reagent.
In one embodiment the dried reagent remains intact until the second or third zones are rehydrated with the fluid sample and a buffer solution.
In one embodiment the dried reagent can be spotted in singular or multiple spots in said second and/or third zones.
In one embodiment the second or third zone comprises multiple dried reagents.
In one embodiment the cuvette comprises a single volume cuvette configured to allow for optical measurement of the buffer solution, the fluid sample and the rehydrated reagents used in each phase of an assay.
In one embodiment the system is configured for performing an immunoturbidimetric or an enzyme-based clinical chemistry assay.
In one embodiment there is provided a sample metering chamber configured to receive the fluid sample and meter a pre-defined volume of the sample and a buffer metering chamber configured to meter a pre-defined volume of buffer solution.
In one embodiment there is provided a sample mixing chamber coupled to the sample metering chamber and coupled to the buffer metering chamber, wherein the sample mixing chamber is configured to mix the sample volume transferred from the sample metering chamber with the volume of buffer solution transferred from the buffer metering chamber to form a dilution of the sample.
In one embodiment there is provided a diluted sample metering chamber coupled between the sample mixing chamber and the reaction chamber, wherein the diluted sample metering chamber is configured to meter a pre-defined volume of the dilution of the sample for transfer to the reaction chamber.
In one embodiment there is provided a reaction chamber coupled to the diluted sample metering chamber.
In one embodiment there is provided two or more reaction chambers each reaction chamber comprising at least the first zone, and wherein at least one reaction chamber has the at least three zones.
In one embodiment there is provided a sample dilution chamber for mixing the fluid sample and a buffer solution, and a distribution channel coupled between the sample dilution chamber and the two or more reaction chambers, wherein the distribution channel is configured to deliver a diluted sample from the sample dilution chamber downstream to each of the two or more reaction chambers in sequence.
In one embodiment there is provided a delivery channel associated with each reaction chamber, wherein the diluted sample is delivered from the distribution channel to each reaction chamber by means of its delivery channel.
In one embodiment there is provided an overflow chamber coupled to the distribution channel for receiving the diluted sample which remains after delivery to the two or more reaction chambers.
In one embodiment there is provided a buffering chamber coupled to the distribution channel, wherein the buffering chamber is configured to prevent cross contamination between two or more of the reaction chambers.
In one embodiment there is provided an intermediate sample metering chamber coupled between one of the reaction chambers and its delivery channel, wherein the intermediate sample metering chamber is configured to prevent cross-contamination between the two or more reaction chambers.
In one embodiment there is provided an intermediate chamber coupled between each delivery channel and its reaction chamber.
In one embodiment each intermediate chamber comprises a metering chamber and an overflow chamber configured such that the metering chamber is filled with diluted sample from the distribution channel until the centrifugal pressure applied to the delivery channel is equal to the pressure in the overflow chamber.
In one embodiment there is provided a buffering chamber coupled to the distribution channel, wherein the buffering chamber comprises a first section and a second section linked by a capillary channel.
In a further embodiment there is provided a microfluidic system comprising:
In another embodiment there is provided, a microfluidic system comprising:
The invention will be more clearly understood from the following description of an embodiment thereof, given by way of example only, with reference to the accompanying drawings, in which:
The POC cartridge can include a buffer reservoir and will have a means to apply a sample (for example whole blood, plasma, serum) to the cartridge. The cartridge may contain dried, immobilised reagents (R1 and R2) stored in specific locations on the cartridge that can be rehydrated independently. Depending on where the sample is added in the sequence (option (a) or (b) in
Referring to
In operation, centrifugal force is used to control the delivery of a stored buffer from its reservoir 10 and/or subsequent buffer chambers prior to being delivered to the reaction chamber 15. The reaction chamber 15 is sized such that it is much greater than the buffer reaction volume that will be used. The reaction chamber 15 incorporates three distinct zones: A) cuvette detection zone, B) R1 reagent zone and C) R2 reagent zone. The cuvette 45 is located at the radial extent of the reaction chamber 15 (typically close to the cartridge outer diameter 20). The chamber extends radially inward on two sides to create two zones that can be independently filled with fluid for the R1 and R2 reactions. It is beneficial that each zone is sized such that when occupied by buffer they can hold the entire volume within the zone, i.e. the volume of zone A, B or C is equal or greater than the buffer volume and the entire reaction chamber 15 is at a minimum of 3× greater than the buffer volume.
Typically it is very difficult to move fluids radially inward using centrifugal microfluidics as the primary means of fluid movement. This can limit/restrict the options available to allow a sequential assay to be performed. To overcome this problem, a combination of centrifugal force and gravity are used to move fluids radially outward and inward respectively. When the cartridge 5 rotates at velocities where the relative centrifugal force (RCF) is much greater than gravity, centrifugal forces will dominate and fluid can be moved radially outward on the cartridge. When the cartridge 5 is stationary or rotating slowly, gravity will still influence the fluid and can be used to move the fluid. To take advantage of this, the cartridge 5 is rotated on an inclined plane (from the horizontal) such that the cartridge 5 can be positioned statically to create a downward slope for fluid to flow. This method can be employed to move fluids radially inward on the cartridge when it is aligned in particular orientations. The flow of fluid under gravity can also be aided by gentle agitation/shaking to overcome any effects of surface tension that may prevent fluids from flowing.
In
The cartridge 5 is then aligned to allow the fluid within Zone A to flow to Zone B under gravity (aided by gentle agitation if required). The sample and buffer suspension wets reagent R1 and begins rehydrating it. The rehydration continues for a defined period of time until full rehydration has been achieved. This rehydration can be aided by mixing/agitation. When fully rehydrated, centrifugal force is used to move the sample, buffer and R1 suspension back to the cuvette 45 (Zone A) where a calibration measurement can be performed on this suspension.
Similar to the rehydration of reagent R1, the cartridge 5 is then orientated to allow the fluid to flow from the cuvette 45 to Zone C where the R2 reagent(s) are wetted by the buffer, sample and R1 suspension. Again, rehydration continues for a defined period of time until both dried reagents are fully rehydrated. The rehydration can again be aided by mixing agitation on the cartridge 5. Finally, the entire fluid volume is returned to the cuvette 45 (Zone A) where the final reaction can be monitored. It is worth noting that reagents R1 and/or R2 can be spotted in singular or multiple spots.
Illustrated in
For example
The cartridge is then aligned to allow the fluid within Zone A to flow to Zone B under gravity (aided by gentle agitation if required). The sample and buffer suspension wets reagent R1 and begins rehydrating it. The rehydration continues for a defined period of time until full rehydration has been achieved. This rehydration can be aided by mixing/agitation. When fully rehydrated, centrifugal force is used to move the sample, buffer and R1 suspension back to the cuvette 45 (Zone A) where a calibration measurement can be performed on this suspension.
Similar to the rehydration of reagent R1, the cartridge is then orientated to allow the fluid to flow from the cuvette 45 to Zone C where the R2 reagents (split in to reagents R2-A and R2-B) are wetted by the buffer, sample and R1 suspension. Again, rehydration continues for a defined period of time until both dried reagents are fully rehydrated. The rehydration can again be aided by mixing agitation on the cartridge. Finally, the entire fluid volume is returned to the cuvette 45 (Zone A) where the final reaction can be monitored. Reagents R1 and/or R2 can be spotted in singular or multiple spots.
In the context of the present invention the term ‘zone’ can be interpreted as an area within a chamber than can be wholly filled with fluid without wetting or filling neighbouring zones within the same chamber. In practice, this means that the volume of fluid used is typically much less than the total volume of the chamber and is only sufficient to occupy a single zone at any given time. The fluid is then manipulated between each zone by centrifugal or gravitational force. Each zone can be further distinguished or protected from neighbouring zones by physical barriers incorporated in the shape and design of the reaction chamber.
The cartridge comprises a reaction chamber 15 having at least three zones. The reaction chamber 15 as shown is substantially oblong in the radial direction but it is understood that the shape can be modified for optimal performance such as elliptical, circular, zig-zag or other desired shape to accommodate the three zones. The reaction chamber 15 may also have additional mechanical features (not shown) to better distinguish the individual zones in operation. For example, the centre of the chamber may have a restriction in width and/or depth in relation to either end of the reaction chamber.
A first zone A is positioned at the radial extent (i.e. furthest from the centre of rotation 25) of the reaction chamber 15 and defines the detection zone containing the cuvette 45 for optical interrogation of fluid. A second zone B is positioned radially inward of Zone A and contains the first reagent spot location R1. A third zone C can be positioned at the most radially inward end of the reaction chamber 15 and contains a second reagent spot location R2. It will be appreciated that the third zone can also be positioned in the same radial position as the second zone if required. The motor and a control module is configured to provide a combination of centrifugal force and gravitational force to move said fluid sample between the three zones.
In operation, centrifugal force is used to control the delivery of a stored buffer from its reservoir 10 and/or subsequent buffer chambers prior to being delivered to the reaction chamber 15. The reaction chamber 15 is sized such that it is much greater than the buffer reaction volume that will be used. The reaction chamber 15 incorporates three distinct zones: A) cuvette detection zone, B) R1 reagent zone and C) R2 reagent zone. The cuvette 45 is located at the radial extent of the reaction chamber 15 (typically close to the cartridge outer diameter 20). The chamber is dimensioned to allow for the creation of two zones that can be independently filled with fluid for the R1 and R2 reactions. It is beneficial that each zone is sized such that when occupied by buffer they can hold the entire volume within the zone, i.e. the volume of zone A, B or C is equal or greater than the buffer volume and the entire reaction chamber 15 is preferably at a minimum of 3× greater than the buffer volume.
Typically it is very difficult to move fluids radially inward using centrifugal microfluidics as the primary means of fluid movement. This can limit/restrict the options available to allow a sequential assay to be performed. To overcome this problem, a combination of centrifugal force and gravity are used to move fluids radially outward and inward respectively. When the cartridge 5 rotates at velocities where the relative centrifugal force (RCF) is much greater than gravity, centrifugal forces will dominate and fluid can be moved radially outward on the cartridge. When the cartridge 5 is stationary or rotating slowly, gravity will still influence the fluid and can be used to move the fluid. To take advantage of this, the cartridge 5 is rotated on an inclined plane (from the horizontal) such that the cartridge 5 can be positioned statically to create a downward slope for fluid to flow. This method can be employed to move fluids radially inward on the cartridge when it is aligned in particular orientations. The flow of fluid under gravity can also be aided by gentle agitation/shaking to overcome any effects of surface tension that may prevent fluids from flowing.
In
The cartridge 5 is then aligned to allow the fluid within Zone A to flow to Zone B under gravity (aided by gentle agitation if required). The sample and buffer suspension wets reagent R1 and begins rehydrating the reagent. The rehydration continues for a defined period of time until full rehydration has been achieved. This rehydration can be aided by mixing/agitation. When fully rehydrated, centrifugal force is used to move the sample, buffer and R1 suspension back to the cuvette 45 (Zone A) where a calibration measurement can be performed on this suspension.
Similar to the rehydration of reagent R1, the cartridge 5 is then orientated to allow the fluid to flow from the cuvette 45 to Zone C where the R2 reagent(s) are wetted by the buffer, sample and R1 suspension. Again, rehydration continues for a defined period of time until both dried reagents are fully rehydrated. The rehydration can again be aided by mixing agitation on the cartridge 5. Finally, the entire fluid volume is returned to the cuvette 45 (Zone A) where the final reaction can be monitored. It is worth noting that reagents R1 and/or R2 can be spotted in singular or multiple spots.
Firstly, the buffer volume can be delivered from the buffer chamber 10 to the reaction chamber 15 where it fills Zone A under centrifugal force. At this point, an optical measure or blank can be taken of the buffer volume in the cuvette 45. Sample is then delivered from the sample chamber 35 to the reaction chamber 15 under centrifugal force where it mixes with the buffer volume already contained in Zone A. A sample measurement can be taken at this point.
The rehydrated reagent R1-X can then be delivered from the rehydration chamber 46 to the reaction chamber 15 to mix with the diluted sample and buffer volume already present in Zone A. The cartridge 5 is then aligned to allow the fluid within Zone A to flow to Zone B under gravity if a secondary R1 reagent is present. This rehydration can be aided by mixing/agitation. When fully rehydrated, centrifugal force is used to move the sample, buffer and R1 suspension back to the cuvette 45 (Zone A) where a calibration measurement can be performed on this suspension.
The cartridge 5 is then orientated to allow the fluid to flow from the cuvette 45 to Zone C where the R2 reagent(s) are wetted by the buffer, sample and R1 suspension. Again, rehydration continues for a defined period of time until both dried reagents are fully rehydrated. The rehydration can again be aided by mixing agitation on the cartridge 5. Finally, the entire fluid volume is returned to the cuvette 45 (Zone A) where the final reaction can be monitored.
Secondly/alternatively, the above sequence can be altered such that the rehydrated R1-X volume can be delivered from the rehydration chamber 46 to the reaction chamber 15 before the buffer or sample. This allows for a reagent blank to be measured optically in the cuvette 45 prior to further dilution with buffer or the addition of sample. The advantages of this method are:
Using centrifugal force, the sample in chamber 35 is delivered to a sample metering chamber 54, where a pre-defined sample volume required for the test is metered. In parallel, centrifugal force is also used to deliver an aliquot of buffer from chamber 10 to a first buffer metering chamber 52. Subsequently, a second aliquot of buffer is delivered to a second buffer metering chamber 53 and the excess of buffer from chamber 10 is delivered to an overflow metering chamber 58. Chamber 58 can be used as a procedural control to determine if buffer has been delivered to chambers 52, 53 and 58.
The buffer siphons in the first buffer metering chamber 52 and the second buffer metering chamber 53 are then primed using an acceleration profile provided by the motor attached to the cartridge at 25. These accelerated primed siphons do not require hydrophilic coatings to function. When the buffer siphons are primed, centrifugal force is used to move the buffer in the first buffer metering chamber 52 downstream to a sample mixing chamber 55 and in parallel move the buffer in the second buffer metering chamber 53 to the rehydration chamber 46. A suction effect is then used to transfer the sample aliquot from the sample metering chamber 54 into the sample mixing chamber 55 after the buffer aliquot from the first buffer metering chamber 52 has been delivered.
Sample and buffer are then mixed in the sample mixing chamber 55 to lyse the sample (for HbA1c) and homogenise the dilution. Other assays require the use of plasma instead of whole blood (as required for HbA1c) and the lysis step would not be required in this case. In parallel to sample mixing, the R1-X reagent in the rehydration chamber 46 is rehydrated by the buffer aliquot from the second buffer metering chamber 53.
The next operation in the cartridge is to prime the siphon exiting the sample mixing chamber 55 (on its left hand side) using an acceleration profile from the motor. This transfers the dilution downstream to the diluted sample metering chamber 56 where an aliquot of this dilution is metered. The excess of this dilution is also transferred to a reaction chamber 57 where it can be used as a procedural control to ensure sufficient sample has been delivered and/or to monitor a reaction after reagent R3 (if required) has been rehydrated.
A final acceleration profile from the motor is used to prime the siphon exiting the diluted sample metering chamber 56 and in parallel the siphon exiting the rehydration chamber 46. Using centrifugal force, the metered volume of the diluted sample from the diluted sample metering chamber 56 and the rehydrated reagent dilution from the rehydration chamber 46 are delivered simultaneously to the reaction chamber 15. This final dilution is then homogenised using a mixing profile from the motor in Zone A and an optical measurement of sample and R1-X is taken from the optical cuvette 45. A secondary reagent R1 in Zone B can also be rehydrated and mixed with this dilution. Similarly reagent R1-X could be placed at R1 and rehydrated in the reaction chamber 15 instead. For a HbA1c test this corresponds to the lysed sample being mixed with latex beads (R1-X and/or R1).
The cartridge 5 is then orientated to allow the fluid to flow from the cuvette 45 (and sitting in Zone A and B) to Zone C where the R2 reagent(s) are wetted by the buffer, sample and R1-X (and/or R1) suspension. Again, rehydration continues for a defined period of time until the reagents are fully rehydrated. The rehydration can again be aided by mixing agitation on the cartridge 5. Finally, the entire fluid volume is returned to the cuvette 45 (Zone A) using centrifugal force where the final reaction can be monitored. For the HbA1c test, this corresponds to the antibody complex reagents being rehydrated by the dilution of the lysed sample and latex beads. This agglutination phase is then optically monitored at cuvette 45.
The buffer volume can be delivered from the buffer chamber 10 to the reaction chamber 15 where it fills Zone A under centrifugal force. At this point, an optical measure or blank can be taken of the buffer volume in the cuvette 45. The sample is then delivered from the sample chamber 35 to the reaction chamber 15 under centrifugal force where it mixes with the buffer volume already contained in Zone A. A sample measurement can be taken at this point.
The cartridge 5 is then aligned to allow the fluid within Zone A to flow to Zone B under gravity where reagent R1 is present and can be rehydrated. This rehydration can be aided by mixing/agitation. When fully rehydrated, centrifugal force is used to move the sample, buffer and R1 suspension back to the cuvette 45 (Zone A) where a calibration measurement can be performed on this suspension.
The rehydrated R2-Y volume can then be delivered from the rehydration chamber 47 to the reaction chamber 15 where it can be mixed with the buffer/sample/R1 suspension already present in the reaction chamber. Mixing of these volumes can be further enhanced by centrifugal or gravitational means before the mixed suspension is returned to Zone A where the endpoint reaction can be monitored in the cuvette 45. The advantage of this embodiment is that the reagent R2-Y can be rehydrated in parallel with other assay processes such as blank measurement, sample/buffer delivery and R1 rehydration, thus reducing the total assay time.
The buffer volume can be delivered from the buffer chamber 10 to the reaction chamber 15 where it fills Zone A under centrifugal force. At this point, an optical measure or blank can be taken of the buffer volume in the cuvette 45. The sample is then delivered from the sample chamber 35 to the reaction chamber 15 under centrifugal force where it mixes with the buffer volume already contained in Zone A. A sample measurement can be taken at this point. In parallel to the above steps, the reagents R1-X and R1-Y have been fully rehydrated in their respective chambers 46 and 47.
The rehydrated reagent R1-X can then be delivered from the rehydration chamber 46 to the reaction chamber 15 to mix with the diluted sample and buffer volume already present in Zone A. The cartridge 5 is then aligned to allow the fluid within Zone A to flow to Zone B under gravity if a secondary R1 reagent is present. This rehydration can be aided by mixing/agitation. When fully rehydrated, centrifugal force is used to move the sample, buffer and R1 suspension back to the cuvette 45 (Zone A) where a calibration measurement can be performed on this suspension.
The rehydrated R2-Y volume can then be delivered form the rehydration chamber 47 to the reaction chamber 15 where it can be mixed with the buffer/sample/R1 suspension already present in the reaction chamber. If present, a secondary R2 reagent can be rehydrated in Zone C at this point. Mixing of these volumes can be further enhanced by centrifugal or gravitational means before the mixed suspension is returned to Zone A where the endpoint reaction can be monitored in the cuvette 45. The advantages of this embodiment are:
As the diluted sample is delivered to each reaction chamber under centrifugal force, Zone A is filled where a sample measurement can be performed in the cuvette 45. The cartridge 5 is then aligned to allow the fluid within Zone A to flow to Zone B under gravity (aided by gentle agitation if required). The sample and buffer suspension wets reagent R1 and begins rehydrating it. The rehydration continues for a defined period of time until full rehydration has been achieved. This rehydration can be aided by mixing/agitation. When fully rehydrated, centrifugal force is used to move the sample, buffer and R1 suspension back to the cuvette 45 (Zone A) where a calibration measurement can be performed on this suspension.
Similar to the rehydration of reagent R1, the cartridge 5 is then orientated to allow the fluid to flow from the cuvette 45 to Zone C where the R2 reagent(s) are wetted by the buffer, sample and R1 suspension. Again, rehydration continues for a defined period of time until both dried reagents are fully rehydrated. The rehydration can again be aided by mixing agitation on the cartridge 5. Finally, the entire fluid volume is returned to the cuvette 45 (Zone A) where the final reaction can be monitored. It is worth noting that reagents R1 and/or R2 can be spotted in singular or multiple spots. The advantage of this embodiment is that a multiplexed assay can be performed on a single cartridge in isolated reaction chambers preventing the risk of cross contamination.
Using centrifugal force, the sample in chamber 35 is delivered to a plasma separation and metering chamber 59, where a pre-defined blood sample volume is first metered. In parallel this centrifugal force is used to deliver an aliquot of buffer from chamber 10 into the first buffer metering chamber 52 and the excess of buffer from chamber 10 is delivered to the overflow metering chamber 58. Chamber 58 can be used as a procedural control to determine if buffer has been delivered to chambers 52 and 58. The centrifugal force is then increased to separate the cellular components from the plasma in the plasma separation and metering chamber 59.
The plasma siphon exiting the plasma separation and metering chamber 59 and the buffer siphon exiting the first buffer metering chamber 52 are then primed using an acceleration profile provided by the motor attached to the cartridge at 25. When the siphons are primed, centrifugal force is used to move the metered plasma from the plasma separation and metering 59 and the metered buffer from the first buffer metering 52 downstream to the sample dilution chamber 51 where the plasma and diluent is mixed.
Once the sample has been diluted and mixed, it is delivered downstream through a distribution channel 48 to each reaction chamber 15, 15A, and 15B and to a buffering chamber 62, which prevents cross-contamination between 15A and 15B, and an overflow chamber 63. It should be understood that two or more separate reaction chambers can be present per cartridge. The diluted sample is delivered to each sequential reaction chamber via individual delivery channels 49, 50 and 60. Between the delivery channel 49 and the reaction chamber 15, there is an intermediate sample metering chamber 61 which is used to prevent cross-contamination between reaction chamber 15, 15A and 15B. The siphon connecting the intermediate sample metering chamber 61 and reaction chamber 15 is primed using an acceleration profile provided by the motor and the metered sample is then delivered to the reaction chamber 15 using centrifugal force.
This diluted sample volume fills cuvette 45 (Zone A) in reaction chambers 15, 15A and 15B respectively and an individual blank measurement can be performed in each. During the diluted sample delivery steps, the centrifugal force ensures that no fluid reaches Zone B or Zone C (in reaction chamber 15 only) and the dried reagents remain intact until R1 and R2 (in reaction chamber 15 only) are to be rehydrated.
The cartridge is then aligned to allow the fluid within Zone A to flow to Zone B under gravity (aided by gentle agitation if required) in reaction chambers 15, 15A and 15B. The diluted sample wets reagent R1 in all three reaction chambers 15, 15A and 15B and begins rehydrating them in parallel. The rehydration continues for a defined period of time until full rehydration has been achieved. This rehydration can be aided by mixing/agitation. When fully rehydrated, centrifugal force is used to move the diluted sample and R1 suspension back to the cuvette 45 (Zone A) where measurements can be performed on these suspensions.
For two-phase reactions that contain a second reagent R2, as shown in reaction chamber 15 only, the cartridge is then orientated to allow the fluid to flow from the cuvette 45 to Zone C where the R2 reagent is wetted by the diluted sample and R1 suspension. Again, rehydration continues for a defined period of time the R2 reagent is fully rehydrated. The rehydration can again be aided by mixing agitation on the cartridge. Finally, the entire fluid volume is returned to the cuvette 45 (Zone A) in reaction chamber 15 where the final two-phase reaction can be monitored. Reagents R1 and/or R2 can be spotted in singular or multiple spots.
Using centrifugal force, the sample in chamber 35 is delivered to the plasma separation and metering chamber 59, where a pre-defined blood sample volume is first metered. In parallel this centrifugal force is used to deliver an aliquot of buffer from chamber 10 into the first buffer metering chamber 52 and the excess of buffer from chamber 10 is delivered to the overflow metering chamber 58. Chamber 58 can be used as a procedural control to determine if buffer has been delivered to chambers 52 and 58. The centrifugal force is then increased to separate the cellular components from the plasma in the plasma separation and metering chamber 59.
The buffer siphon exiting the first buffer metering chamber 52 is then primed using an acceleration profile provided by the motor attached to the cartridge at 25. This accelerated primed siphon does not require a hydrophilic coatings to function. When the buffer siphon is primed, centrifugal force is used to move the metered buffer from the first buffer metering 52 downstream to the sample dilution chamber 51. A suction effect is then used to transfer the plasma volume downstream from the plasma separation and metering chamber 59 into the sample dilution chamber 51 where the plasma and diluent is mixed.
Once the sample has been diluted and mixed, it is delivered downstream through a distribution channel 48 sequentially to a buffering chamber 66, reaction chamber 15, 15A, and 15B and to the sample dilution overflow chamber 63. It should be understood that two or more separate reaction chambers can be present per cartridge. Buffering chamber 66 is placed at the beginning of the distribution channel 48 to ensure non-homogeneous diluted sample flows in here instead of into the reaction chambers 15, 15A and 15B. The buffering chamber comprises a first section 66a and a second section 66b linked by a capillary channel 67. The diluted sample passes through a delivery channel 49 using centrifugal force and into the first intermediate chamber 61 which contains a metering chamber and an overflow chamber. The metering chamber is filled with diluted sample first before the overflow fills and blocks its vent. The pressure in the overflow chamber will then increase and the diluted sample flow through delivery channel 49 will stop when the centrifugal pressure being applied to the delivery channel 49 is equal to the pressure in the overflow chamber. When the diluted sample stops flowing through the delivery channel 49, a second intermediate chamber 64 is filled in the same way through delivery channel 50. A third intermediate channel 65 is filled in the same manner through the delivery channel 60 prior to the remaining diluted sample being transferred to the overflow chamber 63 via the distribution channel 48.
When all of the diluted sample is delivered from the sample dilution chamber 51, the centrifugal force produced by the motor is increased to break the capillary channel 67 in the buffering chamber 66 so that the diluted sample passes radially outward from the first section 66a to the second section 66b. In parallel the centrifugal pressure in delivery channels 49, 50 and 60 will increase and the diluted sample remaining in these channels will be flushed out and the pressure in the overflow chambers of the first, second and third intermediate chambers, 61, 64 and 65 respectively will return to normal atmospheric pressure. This allows the downstream fluidics to operate as expected and ensure the transfer of fluids from 61 to 15, 64 to 15A and 65 to 15B when required.
For two-phase assays, a first reagent R1 can be placed in the first, second and third intermediate chambers 61, 64 and 65 and these dried reagents are rehydrated by the metered volumes of diluted sample. An acceleration profile from the motor is then used to transfer this dilution from the first, second and third intermediate chambers 61, 64 and 65 via their exit siphons to the reaction chambers 15, 15A and 15B downstream. This dilution volume fills cuvette 45 (Zone A) in reaction chambers 15, 15A and 15B respectively and an individual blank measurement can be performed in each. During the dilution delivery steps, the centrifugal force ensures that no fluid reaches Zone B in the reaction chambers 15, 15A and 15B and the dried reagents in Zone B (first or second reagents R1, R2) remain intact until they are to be rehydrated.
The cartridge is then aligned to allow the fluid within Zone A to flow to Zone B under gravity (aided by gentle agitation if required) in reaction chambers 15, 15A and 15B. The dilution wets these reagents in all 3 reaction chambers 15, 15A and 15B and begins rehydrating them in parallel. The rehydration continues for a defined period of time until full rehydration has been achieved. This rehydration can be aided by mixing/agitation. When fully rehydrated, centrifugal force is used to move this dilution back to the cuvette 45 (Zone A) where measurements can be performed on these suspensions.
will be appreciated from the above description that microfluidic system of the present invention is suitable for performing any type of immunoturbidimetric and enzyme-based clinical chemistry assay. Furthermore, the microfluidic system of the present invention is very flexible, as it can be used to perform an assay that requires the addition and rehydration of a single reagent, as well as to perform an assay that requires the addition and rehydration of multiple reagents. This is due to the fact that the second and/or third reagent zones of the cartridge can each be provided with multiple reagent spots.
In the specification the terms “comprise, comprises, comprised and comprising” or any variation thereof and the terms include, includes, included and including” or any variation thereof are considered to be totally interchangeable and they should all be afforded the widest possible interpretation and vice versa.
The invention is not limited to the embodiments hereinbefore described but may be varied in both construction and detail.
| Number | Date | Country | Kind |
|---|---|---|---|
| 1806931.0 | Apr 2018 | GB | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2019/060885 | 4/29/2019 | WO | 00 |
