Field of Invention

The invention relates to a microfluidic loading module and to a microfluidic loading method for the loading sample into a microfluidic process flow cell module, and for example an analyser such as a genomic sequencer, of a collected and prepared sample of target material such as biological material in a liquid medium for example in solution or suspension therein.

The invention in particular relates to the loading into a process flow cell for further processing and/ or analysis of a target material previously collected from an environment and into a liquid medium for example by a suitable microfluidic sample collection module for subsequent processing and analysis, and in particular for molecular biological analysis such as genomic analysis.

The invention thus in particular relates to a microfluidic loading module intended to sit fluidly between a microfluidic sample collection module and a microfluidic process flow cell module to effect fluid transfer of sample collected by the microfluidic sample collection module to the microfluidic process flow cell module for further processing and for example analysis and for example molecular biological analysis such as genomic analysis.

Background

The ability to analyse biological samples, and in particular environmentally collected biological samples, to identify the presence of particular biological marker, might have a range of applications, for example including include the detection of environmental biological hazards, monitoring and/or control of pollution, monitoring and/or control of airborne pathogens and the like. The ability to identify specific target biological hazards may have particular value in this regard.

Laboratory based technology for analysis of biological samples is relatively well established. Known analysis techniques include genomic sequencing of material isolated from a biological sample. Laboratory based technology for processing and preparation of biological samples to isolate and prepare genomic material for analysis is also relatively well established.

For laboratory application, a batch processing methodology is typically followed where samples are collected in the field and sent to the laboratory for processing and preparation and for subsequent analysis.

Laboratory based technology for subsequent processing and analysing of biological samples using microfluidic lab-on-chip principles finds increasing application. The use of suitable microfluidic process flow cell modules for further processing and for example analysis and for example molecular biological analysis such as genomic analysis of collected samples of biological material in a liquid medium is of increasing significance as a means of automating the process and producing rapid and reliable results.

Advantages can also accrue if the sample collection and where applicable preliminary processing operations can be at least partly automated and reduced in scale, for example allowing it to be performed at least partly at a distributed location, and for example deployed in a portable manner. This may particularly be the case in relation to the analysis of biological samples collected in the field, and for example from the air at a monitored location or site, to detect the presence of biological threats.

For example, patent application No PCT/GB2021/050575 describes a sample collection module which may be amenable to microfluidic principles for the collection of material from a gas stream, and for example from air, that might be particularly suited to application to collect biological samples from the air in a sampling location into an aqueous buffer.

For example, patent application No PCT/GB2022/050889 describes a sample preparation module which may be amenable to microfluidic principles and which may be used as part of a sample collection module or system for the automation of sample collection and preliminary processing operations to prepare a sample for analysis. Microfluidic sample collection modules applying on these and other principles might be sued to collect samples, such as biological samples, into a liquid medium for subsequent processing and analysis, and in particular for molecular biological analysis such as genomic analysis, using suitable microfluidic process flow cell modules as above mentioned.

Typically, the sample priming of standard known microfluidic process flow cell modules, and for example to standard known microfluidic process flow cell modules for genomic analysis, using collected sample material is effected manually. In order to create a more automated sequencing based detection system a microfluidic loading module needs to be developed that is capable of loading a prepared sample into such flow cell. In particular in cases where the collection of a prepared sample is also automated it is desirable to develop a microfluidic loading module intended to sit fluidly between a microfluidic sample collection module and a microfluidic process flow cell module to effect this.

A process flow cell module that has been traditionally designed for use by scientists with manual pipettes is not ideal for integrating into an automated system and the design of such a microfluidic loading module is not straightforward. While the problems with conventional systems may vary with precise design and loading protocols, in a typical process flow cell designed for sequencing they may include some or all of the following.

1. The protocol for taking a flow cell (new or otherwise) and priming it is often heavily dependent on visual feedback and tactile pipette control, requiring the user to adjust their actions based on what they see occurring in the flow cell.

2. A typical flow cell has three ports through which the sample (fluid) and a selection of reagents (fluid) need to be added and removed in a certain order to enable the sequencing or process to work. The ports on the flow cell have been designed to accept reagents from a manually operated pipette which therefore are not of the correct form/fit to take a standard automated connection such as a tube or thread. A typical flow cell design may include a priming port through which a priming mix is added in a first preparatory stage; a sample port through which a reagent mix including the sample may be loaded in a second preparatory stage; and a waste port through which reagents may be removed during operation of the flow cell. Typically, sample is loaded in a dropwise fashion, for example using a pipette or device with a similar action. Such sample ports are for example referred to as a "spot-on port" in certain known process flow cell modules.

3. Sequencing arrays may be susceptible to introduced air bubbles introduced into the sequencing array. Individual sequencing pores that the bubble comes in contact with may be permanently damaged/degraded. Even one small air bubble moving across the array may damage multiple pores which then cannot be used for sequencing. Therefore it is critical that no air bubbles are introduced into the flow cell as it significantly affects the reusability of the flow cell.

4. The sample may typically need to be pushed from the upstream processes. Extra fluid can’t be used to push the sample on to the flow cell as this would dilute the sample and/or this extra fluid could interfere with the function of the flow cell. Therefore air is often used to push the fluid sample along the tube, however only the fluid sample can be allowed to enter the flow cell, the air that is in front of and behind the sample in the tube must be prevented from entering the flow cell (for reasons described above).

5. The protocol for loading a sample onto the flow cell often relies on the user adding a single drop of sample at a time onto the sample port and waiting for that drop to be wicked into the sequencing array before adding another drop (a sample consists of multiple drops). If a new drop is added before the previous has been absorbed into the flow cell array then the new drop overflows and cannot be sequenced, thus the DNA is lost.

6. After a sample has been sequenced the waste material needs to be removed from the flow cell without introducing air into the flow cell to allow the next sample to be loaded. Any automated loading module needs to be capable of removing waste material without introducing air (for reasons described above). 7. The interface between flow cells and the automated loading module must enable easy the flow cells to be easily replaced as they are consumable devices.

It is desirable to develop a microfluidic loading module that mitigates some or all of the above problems, and that is thus adapted to facilitate a more automated loading of a microfluidic process flow cell.

It is in particular desirable to develop a microfluidic loading module that is thereby adapted to sit fluidly between a microfluidic sample collection module and a microfluidic process flow cell module to effect a more automated loading of a sample from the former into the latter. More completely it is desirable to provide a system comprising such a microfluidic sample collection module, microfluidic loading module, and microfluidic process flow cell module fluidly in series.

Summary of Invention

According to the invention in a first aspect, a microfluidic loading module operable in use in fluid communication with a microfluidic process flow cell module having a sample port, a priming port and a waste port is provided comprising: a sample inlet adapted to receive a sample liquid of a target material such as a biological material in a liquid medium; a sample port interface including a sample outlet adapted to effect fluid communication with a sample port of the microfluidic process flow cell module; a sample flow conduit between the sample inlet and sample outlet, including a sample valve operable selectively to open and close a flow path between the sample inlet and the sample outlet; a priming inlet adapted to receive a priming buffer liquid; a priming port interface including a priming outlet adapted to effect fluid communication with a priming port of the microfluidic process flow cell module; a priming flow conduit between the priming inlet and priming outlet, including a priming valve operable selectively to open and close a flow path between the priming inlet and the priming outlet; a waste outlet; a waste port interface including a waste inlet adapted to effect fluid communication with a waste port of the microfluidic process flow cell module; a waste flow conduit between the waste inlet and the waste outlet, including a waste valve operable selectively to open and close a flow path between the waste inlet and the waste outlet; wherein the sample outlet comprises a microfluidic tube having a longitudinal direction and an end profile angled away from a direction transverse to the longitudinal direction.

The loading module is a microfluidic device in particular in that all flow conduits and valves are microfluidic elements. The microfluidic loading module is designed to interface with and automate delivery of sample and buffer to and removal of waste from a microfluidic process flow cell module having a sample port, a priming port and a waste port of the type which will be familiar. To that end, at its most general, it has a sample port interface, a priming port interface and a waste port interface each adapted to effect a fluid communicating engagement with the respective ports on the microfluidic process flow cell and a suitable arrangement of conduits and valves including at least those above identified.

In typical process flow modules, the sample port is provided for loading of a sample into the microfluidic process flow cell module with which the loading module of the invention is intended to operate. Typically, sample is loaded in a dropwise fashion, for example using a pipette or device with a similar action. A distinctive feature of the invention is the provision at the sample outlet of a microfluidic tube having a longitudinal direction and an end profile angled away from a direction transverse to the longitudinal direction. This structure enables the module of the invention to mimic the manual method of loading the flow cell to add the sample to the port one drop at a time.

In a possible embodiment, the sample outlet of the sample port interface comprises a cylindrical microfluidic tube having an end cut at an oblique angle to its long direction to give an end profile angled away from a direct transverse cut.

In a possible embodiment, the cylindrical microfluidic tube has an end cut at an oblique angle to its long direction to give an end profile angled away from a direct transverse cut with desired angle being between 5 and 30 degrees. The angled profile on the end of the tube is key to allowing the liquid to form a drop and go into the flow cell but also allow any air in the sample to escape. With this key feature, in combination with control of the distance between the end of the tube and the flow cell sample port (close enough that small volumes of liquid can bridge the gap between the end of the tube and the sample port but far enough away that air isn’t introduced into the flow cell) provides an effective mimicry of the manual pipette action for which the microfluidic process flow cells to which the invention is intended to engage is designed. A solution is offered in which a microfluidic loading module can be engaged with such microfluidic process flow cells and provide an automated loading solution.

The priming and waste port interfaces may use any simple fluidly sealed engagement to seal with the respective ports. For example simple o-rings may be provided.

The foregoing arrangement of microfluidic inlets, outlets, flow paths and valves arrangement provides the minimum necessary within the microfluidic loading module to interface with and automate delivery of sample and buffer to and removal of waste from a microfluidic process flow cell module having a sample port, a priming port and a waste port.

More complex arrangements of microfluidic flow within the microfluidic loading module may be provided to enhance functionality without departing from the principles of the invention, in particular including those below in any combination. Moreover, reference in the singular to any inlet, outlet, flow path, valve or other component will be understood to include the plural, and in particular plural flow paths in series or in parallel as dictated by the requirements of the function may be provided for any feature of the invention.

In a particular preferred refinement there is provided a second waste flow conduit in fluid communication between the waste outlet of the loading module and the sample valve, and the sample valve comprises a valve to effect selective switching of flow between: a flow path between the sample inlet and the sample outlet and a flow path between the sample inlet and the waste outlet. With this additional flow option, the valve is operable to allow a flush to be pushed through the sample input line and out to waste without affecting the other operations of the loader.

Further optionally, the loading module may comprise: a flush inlet adapted to receive a flush buffer liquid; a flush flow conduit between the flush inlet and the sample outlet, including a flush valve operable selectively to open and close a flow path between the flush inlet and the sample outlet/ sample port interface.

Further optionally, the loading module may comprise: a second priming inlet adapted to receive a priming buffer liquid; a second priming flow conduit between the second priming inlet and the sample outlet, including a second priming valve operable selectively to open and close a flow path between the second priming inlet and the sample outlet/ spot on port interface.

This allows for the dispensing of priming fluid to the sample port of the process flow module. The second priming inlet may be the same as or fluidly parallel to or discrete from the priming inlet supplying the priming port interface.

Further optionally, the loading module may comprise: a push fluid inlet adapted to receive push fluid; a push fluid conduit between the push fluid inlet and the sample outlet, including a push valve operable selectively to open and close a flow path between the compressed air inlet and the sample outlet/ sample port interface.

This may apply a push fluid to the sample and reagents to ensure as much liquid as possible is pushed into the flow cell. The push fluid may be compressed air.

Further optionally, the loading module may comprise: a drawing means such as a syringe pump to draw fluid from the system; a drawing conduit in fluid communication at least with the waste outlet of the loading module and/ or with the waste inlet of the loading module. The drawing means may be used selectively to draw waste from a waste reservoir of the flow cell in use.

In a more complete system, the microfluidic loading module of the first aspect of the invention is provided fluidly coupled to a microfluidic process flow cell module having a sample port, a priming port and a waste port.

In a more complete system, the microfluidic loading module of the first aspect of the invention is provided fluidly coupled between a microfluidic sample collection and processing module and a microfluidic process flow cell module having a sample port, a priming port and a waste port. A suitable microfluidic sample collection and processing module may for example include any of the features described in applicant’s patent application Nos PCT/GB2021/050575, PCT/GB2022/050889.

In a further aspect of the invention, a method is described herein for the loading sample into a microfluidic process flow cell module, and for example an analyser such as a genomic sequencer, of a collected and prepared sample of target material such as biological material in a liquid medium for example in solution or suspension therein.

The method in particular comprises the steps described herein for the operation of the first aspect of the invention, and is for example the use of a device of the first aspect of the invention to effect the same.

For example, a method for the loading sample into a microfluidic process flow cell module comprises: providing a microfluidic loading module in accordance with the first aspect of the invention, in particular having at least: a sample inlet adapted to receive a sample liquid of a target material such as a biological material in a liquid medium; a sample port interface including a sample outlet adapted to effect fluid communication with a sample port of the microfluidic process flow cell module; a sample flow conduit between the sample inlet and sample outlet, including a sample valve operable selectively to open and close a flow path between the sample inlet and the sample outlet; a priming inlet adapted to receive a priming buffer liquid; a priming port interface including a priming outlet adapted to effect fluid communication with a priming port of the microfluidic process flow cell module; a priming flow conduit between the priming inlet and priming outlet, including a priming valve operable selectively to open and close a flow path between the priming inlet and the priming outlet; a waste outlet; a waste port interface including a waste inlet adapted to effect fluid communication with a waste port of the microfluidic process flow cell module; a waste flow conduit between the waste inlet and the waste outlet, including a waste valve operable selectively to open and close a flow path between the waste inlet and the waste outlet; wherein the sample outlet comprises a microfluidic tube having a longitudinal direction and an end profile angled away from a direction transverse to the longitudinal direction; fluidly connecting the microfluidic loading module to a microfluidic process flow cell module having a sample port, a priming port and a waste port such that the sample port interface of the loading module is fluidly connected to the sample port of the flow cell module, the priming port interface of the loading module is fluidly connected to the priming port of the flow cell module, and the waste port interface of the loading module is fluidly connected to the waste port of the flow cell module; supplying sample fluid and priming fluid to the microfluidic loading module; selectively operating the valves of the microfluidic loading module to supply sample fluid and priming fluid to and remove waste from the flow cell module; thereby effecting a desired process in the flow cell module.

In preferred embodiments of all aspects of the invention, the process flow module is adapted to perform and the desired process is an analysis and for example molecular biological analysis such as genomic analysis. In particular the process flow module is preferably a genetic sequencer and the process is sequencing.

Brief Description of Drawings

The invention will now be described by way of example only with reference to the accompanying drawings in which: Figure 1 shows in perspective view a microfluidic chip assembly including a loading module and for performing a loading method in accordance with an example embodiment of the invention;

Figure 2 is an example chip fluid schematic for a loading module and for performing a loading method in accordance with an example embodiment of the invention;

Figure 3 shows in perspective view example interfaces between a microfluidic chip assembly in accordance with an embodiment of the invention and a flow cell;

Figure 4 shows in perspective view a sample cooler for possible use in conjunction with a microfluidic chip assembly in accordance with the invention.

Detailed Description of Drawings

A microfluidic chip assembly including a loading module and for performing a loading method in accordance with an example embodiment of the invention is shown in Figure 1 and a schematic flow diagram for the same is shown in Figure 2. The example embodiment uses a microfluidic chip that is in use clamped to the top of a microfluidic flow cell, which is in particular adapted for analysis of the sample and in the discussed embodiment is a sequencer. The flow cell envisaged for use with the microfluidic chip assembly of the invention is of a design to which reagents are conventionally added manually, for example dropwise using a pipette. A source of sample for the flow cell is a source of a biosample in liquid, for example a microfluidic sample collector/ preparation module, and is in the discussed example a bioreactor. The chip assembly manages the addition of reagents and sample and removal of waste liquids by using a selection of microfluidic valves and sensors and thus facilitates the automation of the process of supplying a prepared sample to the flow cell for analysis and in the example for sequencing.

The flow cell of suitable design may be located in a suitable carrier. The microfluidic loader chip assembly shown in Figure 1 is clamped fluidly to the flow cell, for example on top of the carrier, using a spring loaded clamp that ensures that the correct force is applied between the microfluidic chip and the flow cell to seal the priming and waste ports on the flow cell to the ports in the chip. There is a locating shroud on the bottom of the chip assembly to ensure that the ports on the chip align with the respective ports on the flow cell. The top of the chip assembly consists of the microfluidic chip and valves which control the reagent dispensing and waste aspiration to ensure the correct reagents get dispensed into the correct ports at the correct time.

An example fluidic schematic for the microfluidic loader chip, showing a suitable arrangement of microfluidic flow channels and valves, is shown in Figure 2, and an example mode of operation is discussed.

A flow cell for use with the chip comprises in conventional manner a sample port, a priming port and a waste port. The flow cell is not as such shown in Figure 2, but flow outlets from the chip to these respective ports are identified. The chip further comprises inlets/ sources for a collected and prepared sample supplied by a bioreactor, for a priming buffer, for a compressed air supply, and for a DNase flush; and a waste reservoir/ outlet.

A sample flow channel including sequentially as shown a source channel, valve V1 , and sample port channel, provides a flow path that fluidly connects the prepared sample supply from the bioreactor to the sample port flow outlet to enable supply of sample to the sample port of the flow cell, referred to in the illustrated embodiment as the spot-on port. Valve V1 controls this flow by being operable selectively to open and close the flow path to the flow cell spot-on port. In the example, V1 is a 3/2 valve also connected to a flow channel that provides a flow path that fluidly connects the valve to the waste outlet of the loader chip and is operable to switch the output from the bioreactor between the waste outlet of the loader chip and the flow cell spot-on port. This valve allows a flush of the main system to be pushed through the sample input line to the loader and out to waste without affecting the other operations of the loader. This design provides as large a window as possible for flushing the bioreactor and sample line with the hopes of reducing the down time required to flush the full system.

When a sample is ready for sequencing the valve V1 is switched to supply the spot- on port and the sample pushed into the loader, through the valve and into the flow cell spot on port.

V2 is a 2/2 valve for dispensing the DNase flush buffer into the spot-on port to flush the flow cell between samples. This valve is located upstream of V1 in the spot-on port channel so that the DNase flush also flushes any residue sample out of the spot-on port channel thus reducing carryover.

V3 is 2/2 valve for dispensing the flow cell priming mix into the spot-on port prior to loading a sample. This valve is upstream of V2 in the spot-on port channel so that the priming mix can flush out any DNase flush residue from the spot-on port channel that may degrade the sample.

V4 is another 2/2 valve and is the final valve in the spot-on port channel and is used to provide a small extra air push behind the sample and reagents to ensure as much liquid as possible is pushed into the flow cell.

V5 is a 2/2 valve for dispensing priming mix into the priming port of the flow cell to prepare it for a new sample and is located in a priming port channel that defines a priming flow path between the priming source and the priming port flow outlet of the chip.

V6 is a 3/2 valve which connects an external custom syringe pump to either V7 or the flow cell priming port channel. When V6 is connected to the flow cell priming port it allows the syringe pump to aspirate any air that is in the flow cell priming port and the priming port channel drawing the storage buffer that the flow cells contain when they’re shipped up into the microfluidic chip channel. A sensor is used to detect when the storage buffer has been drawn past V5 which ensures that no air is pushed into the flow cell during priming. The length of the priming port channel has been kept as short as possible to reduce the volume of liquid that has to be drawn back from the flow cell as there is a finite volume of storage buffer in the flow cells. The channel volume is small enough to ensure that the channel is primed before enough storage buffer is drawn out of the flow cell to risk introducing air into the sequencing array.

V7 is another 3/2 valve that connects the syringe pump to either the chip waste outlet or the flow cell waste port. This valve (in combination with V6) allows the syringe pump to draw waste out of the flow cell and then push it out to the system waste reservoir.

Figure 3 shows in perspective view example interfaces between a microfluidic chip assembly in accordance with an embodiment of the invention and a flow cell with the sample or spot-on port interface to the left and the priming port and waste port interfaces to the right. The sample or spot-on port interface comprises a cylindrical microfluidic tube having an end cut at an oblique angle to its long direction to give an end profile angled away from a direct transverse cut, for example by about 5 to 30 degrees.

The angled profile on the end of the tube is key to allowing the liquid to form a drop and go into the flow cell but also allow any air in the sample to escape. The distance between the end of the tube and the flow cell spot-on port is also important as it has to be close enough that small volumes of liquid can ridge the gap between the end of the tube and the spot-on port but far enough away that any air isn’t introduced into the flow cell. This structure enables the tube of the invention effectively and efficiently to mimic the manual method of loading the flow cell by adding sample to the spot-on port one drop at a time.

The priming and waste port interfaces simply use o-rings to seal on the surface around ports on the flow cell.

The waste port interface includes a sensor to detect when the waste reservoir in the flow cell is almost empty as drawing too much of the fluid out of the waste reservoir can potentially cause air to be drawn into the sequencing array through the spot-on port or prevent the next sample from loading properly.

An optional reagent cooler has been designed to prolong the life of the reagents and is shown in Figure 4. The reagent cooler uses a peltier module to keep the reagent reservoirs at 4 to 12°C, as per good practice for storage of proteins.

When located between a source supply module such as the discussed bioreactor and a flow cell module such as the discussed sequencer, a loader in accordance with the principles of the invention facilitates the automation of the process of supplying a prepared sample to the flow cell for analysis and in the example for sequencing. In particular, it facilitates at least in part the automation of the process of adding sample to the spot-on port of a flow cell that is usually configured to receive sample manually, by more effectively mimicking the manual pipette action. Advantages of efficiency and consistency are offered over a manual protocol based on a manually operated pipette. An effective adaptation is made to reduce likelihood of introduced air bubbles which may damage the sequencing array of a flow cell. By operation of the valves and sensors as above, the chip offers an effective automated microfluidic solution to manage the addition of reagents and sample to and removal of waste liquids from the flow cell.