GB2618841A

GB2618841A - Processing module and method

Info

Publication number: GB2618841A
Application number: GB2207403.3A
Authority: GB
Inventors: Smith De Diego Africa
Original assignee: Kromek Ltd
Current assignee: Kromek Ltd
Priority date: 2022-05-20
Filing date: 2022-05-20
Publication date: 2023-11-22
Also published as: GB202207403D0

Abstract

A concentrator module comprises an inlet, at least two outlets, at least one further outlet, and a fluid pathway therebetween including a processing unit that is operable to preferentially divert target biological material to one of the outlets, the processing unit includes elements disposed to operate on the principles of dielectrophoresis (DEP) and alternating current electroosmosis (ACEO). Buffer medium/ undesired solid material/ non-target biological material may be diverted to the further outlet, to be discarded or further processed. The ACEO device and the DEP device may be placed consecutively on the same side of the microfluidic channel. The ACEO may generate a vortex flow to pull particles from the bulk liquid and the DEP may be used to trap particles. Another concentrator module comprises an ACEO electrode system disposed at an angle (e.g. slanted) to the flow direction.

Description

PROCESSING MODULE AND METHOD

Field of Invention

The invention relates to a processing module and method for the concentration of collected biological samples in a buffer fluid for subsequent processing and analysis, and in particular for molecular biological analysis. In preferred embodiments, the invention concerns the analysis of environmentally collected biological samples, for example of airborne biological material which has been collected into a suitable aqueous buffer.

Background to the Invention

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.

Advantages can accrue if the analysis process 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.

Such environmentally collected biological samples may have a wide range of varied biological material present that it is desirable to screen and test via a broad spectrum analysis and in particular a genomic analysis to detect the presence of various specific target sequences identifying specific target biological hazards or pathogens. Collection of sufficient concentrations of any target material for that subsequent analysis is likely to require the collection of relatively large quantities of biological material, for example from large volumes of air, into a suitable buffer fluid such as an aqueous buffer.

Where collection takes place for example from the air at a monitored location or site, to detect the presence of biological threats, particular advantages may accrue if the at least partly automated analysis process can be provided in line with the collection system. The effective collection and concentration of a sample might be particularly important in relation to such in line analysis. However, the large volumes of biological material in buffer fluid that are likely to be collected in the above described scenario do not readily lend themselves to processes involving miniaturised and automated processing systems.

For example, UK patent application No 2003303.1 describes a sampler 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 particular environment into an aqueous buffer. This system produces large volumes of buffer with biological material. The concentration of this material is likely to be a critical early process step in adapting this for use with at least partly miniaturised and automated processing systems. Concentration targets of at least 50 times by volume and possibly 1000 times or more may be desired.

Advantages can accrue if at least some of the subsequent processing apparatus can be made portable and compatible with such a sampler, and for example made able to operate in-line downstream of such a sampler, for example for deployment in distributed environments. Particular advantages can be envisaged if the collection and processing apparatus can be deployed on a vehicle. This allows the ready collection of multiple samples from multiple locations and their rapid analysis, for example for the detection of biological threats present in the sampled environment. However, the volumes of biological material in buffer fluid that are likely to be collected make this more difficult.

The invention in particular concerns a module and method for the concentration of biological samples for subsequent processing for analysis.

The invention in particular concerns a module and method for the concentration of biological samples for subsequent processing that might facilitate the automation of at least some of the stages involved in the processing and analysis of such biological samples, and/ or the facilitation of the performance of those stages away from the laboratory.

The invention in particular seeks to provide a module and method for the concentration of biological samples that is suitable for use in association with a system and method for the processing and preparation of biological samples for analysis and/ or suitable for use in association with a sampling system that can sample an environment for biological material, in particular to collect airborne biological material, and which is in particular suitable for use directly in-line with such a sampling system and processing system allowing for operation in the environment to be sampled, for example in a continuous manner, to detect in real time and in situ the presence of particular target biological hazards or pathogens in the sampled environment.

In the general case, a relatively large volume of collected material/ fluid buffer will be introduced to the module of the first aspect of the invention. At the concentration stage this is reduced into a much smaller volume of liquid (for example to enable it to be more easily further processed). Concentration targets of at least 50 times by volume and possibly 1000 times or more may be desired. For example, in the discussed preferred application of the invention for use with the air sampler described in Applicant's UK patent application No 2003303.1 particulate material in a 45 mililitre collection buffer is ideally concentrated down to 600 microlitres.

The concentrator module must ensure that biological material, and in particular biological material from which DNA/ RNA may be extracted, is retained, but both a substantial proportion of buffer fluid and as much undesired solid material as possible is removed to produce an output in which the biological material is substantially concentrated relative to carrier buffer solution, for example to a factor of at least 50 times by volume relative to input concentration, more preferably at least 500 times, and possibly 1000 times or more.

The desire is for a methodology and system that capable of concentrating a range of biological material from a metagenomic environmental sample for testing, to give a broad spectrum test option. The desire is for a methodology and system that is susceptible for automation/ miniaturization/ use in the field. Conventional solutions do not necessarily meet these desired. Filters may block desired material and would be consumable. Centrifuges are too big. Chromatography columns are expensive.

Other solutions may be targeted rather than broad spectrum.

Particular solutions may be sought in which the concentrator module concentrates cellular material such as bacterial material from a metagenomic sample and/ or concentrates viral particles from a metagenomic sample and/ or concentrates and separates for separate processing both bacterial material and viral material from a metagenomic sample.

Summary of Invention

Solutions are offered in which the principles of dielectrophoresis (DEP) and/ or alternating current electroosmosis (ACEO) are used in various combination in a microfluidic module to effect concentration and/ or separation of one or more particular target materials from a metagenomic environmental sample for subsequent testing.

An advantage of microfluidic systems is the ability to multiplex through multiple channels embodying different processes. This is exploited in that the solutions of the invention module include one or more microfluidic DEP devices and/ or one or more microfluidic ACEO devices which are appropriately configured in conjunction with arrangements of microfluidic channels to effect concentration and/ or separation of one or more particular target materials from the sample. In particular, the solutions herein described provide microfluidic arrangements capable of use with a sample comprising broad spectrum environmentally collected biological material in a fluid buffer medium to concentrate and separate for separate processing both bacterial material and viral material from the sample.

In all cases except where express wording or context demands otherwise where reference is made to the singular it includes the plural and in particular where reference is made to a processing unit including a microfluidic DEP device and a microfluidic ACE0 device such a unit may include more than one such device in appropriate combination, where reference is made to a flow channel or to a device or reactor therein this will encompass the use of multiple channels and in particular of multiple channels in parallel with an equivalent functionality, and where reference is made to inlets and outlets this will encompass the use of multiple inlets and outlets in appropriate fluid communication.

In accordance with the invention in a first aspect a concentrator module to receive a sample comprising biological material in a fluid buffer medium and increase the concentration of a target biological material present within the sample comprises a microfluidic apparatus including: an inlet, an output outlet, at least one further outlet, and a fluid pathway therebetween including a processing unit that is operable to preferentially divert the target biological material to the output outlet, and for example preferentially diverts buffer medium/ undesired solid material/ non-target biological material to the further outlet, for example to be discarded or further processed; wherein the processing unit includes elements disposed to operate on both of the principles of dielectrophoresis (DEP) and alternating current electroosmosis (ACEO).

That is to say, a suitable processing unit includes a microfluidic DEP device and a microfluidic ACE0 device.

In a preferred case, the processing unit comprises at least an upstream microfluidic ACE0 device and a downstream microfluidic DEP device.

By analogy, a method to process a sample comprising biological material in a fluid buffer medium and increase the concentration of a target biological material present within the sample comprises passing the biological material and fluid buffer medium through a concentrator module in accordance with the first aspect to increase the concentration of the biological material.

In particular preferably, the method comprises passing the sample of biological material and fluid buffer medium successively through at least a microfluidic ACE° device and a microfluidic DEP device downstream of the ACE° device.

Applicant's concentrator module of the first aspect of the invention is intended to provide a microfluidic processing unit to effect the desired concentration discussed above, and to tend to preferentially segregate and concentrate larger material, for example over 1 micron in size, and for example bacteria, which can then be separated into a first process flow stream to be diverted to the output outlet. Other material, including the fluid medium and smaller biological material, may be further separated/ concentrated into other process flow stream(s) to be diverted to the further outlet(s) for further processing or discharge as desired.

Thus, in accordance with the invention of the first aspect, a typical target biological material may comprise bacterial material. In other embodiments a typical target biological material may additionally or alternatively comprise viral material.

The first aspect of the invention achieves this objective by the use of both DEP and ACED functionalities integrated into a microfluidic concentrator module.The key to the invention is that both functionalities are exploited in the same microfluidic concentrator module. An advantage of microfluidic systems is the ability to multiplex through multiple channels embodying different processes. This is exploited in that the concentrator module includes one or more microfluidic DEP devices and one or more microfluidic ACED devices.

In a preferred case, the processing unit of the concentrator module comprises at least a microfluidic ACED device and a microfluidic DEP device downstream of the ACED device. Conveniently, the processing unit of the concentrator module comprises a first series of microfluidic pathways each configured for the passage of fluid through one or more microfluidic ACED devices and a second series of microfluidic pathways each configured for the passage of fluid through one or more microfluidic DEP devices. Conveniently, the one or more microfluidic DEP devices are positioned downstream of the one or more microfluidic ACED devices with the first series of microfluidic pathways and the second series of microfluidic pathways forming fluidly continuous pathways successively therethrough.

In preferred embodiments, the microfluidic ACED device comprises an ACED electrode system disposed in parallel to a flow direction through the device, and for example comprises an ACED electrode system disposed on one or more sides of a flow channel in the device.

In preferred embodiments, the processing unit of the concentrator module comprises an upstream microfluidic ACED device having an electrode system configured to be operable to tend to draw biological material flowing through a channel in the device towards one or more sides of the channel and a downstream microfluidic DEP device having an electrode system juxtaposed therewith and operable to tend to act on larger particles thereby drawn into its vicinity.

For example, the invention comprises a microfluidic ACED device having an electrode system disposed in parallel to a flow direction through the device on one or more sides of a flow channel in the device and configured to be so operable.

For example, a microfluidic ACED device having a parallel ACED electrode system and a microfluidic DEP device having a DEP electrode system are positioned fluidly consecutively on the same side of a microfluidic channel.

In operation in accordance with the method of the first aspect of the invention, two stages of operation are employed, comprising: a capture phase in which: the ACED electrode system is operated to tend to cause biological material within the sample to be urged towards the electrode system; the DEP electrode system is operated with positive DEP to trap larger particles of the biological material drawn into its vicinity by the ACED electrode system; material not so captured flows through and beyond both electrode systems and a release phase in which: the DEP electrode system is not operated or is operated with negative DEP to release the trapped larger particles.

In this way, larger particles, for example bacteria, may be preferentially segregated from an input flow, the method producing a first output flow during the capture phase from which larger particles such as bacteria tend to be removed, but smaller particles for example below 1 micron in size and including viruses not affected by the DEP, tend to remain, and a second output flow during the release phase in which larger particles such as bacteria are now separated and concentrated.

Optionally, a flow channel within the microfluidic concentrator module may divide downstream of the microfluidic ACED device to define two onward flow channels, a first onward flow channel being configured such that it receives a flow from that part of the undivided channel in the vicinity of the ACE° electrode system (and into which the biological material predominantly is urged) and a second onward flow channel being configured such that it receives a flow from away from the vicinity of the ACED electrode system. The first onward flow channel may pass into the microfluidic DEP device as above described and the second onward flow channel may pass to discharge.

Thus, in some embodiments, the microfluidic concentrator module may comprise: an inlet flow channel in fluid communication with a microfluidic ACE° device comprising one or more microfluidic channels defining an initial flow direction through the device and an ACEO electrode system disposed in parallel to the flow direction, and operable to tend to cause biological material to segregate into a first part of the flow channel; a divide in the inlet flow channel into a first onward flow channel dividing from the same in such manner as to be fluidly continuous with the first part of the flow channel, and a second onward flow channel; the second onward flow channel optionally continuing to a discharge outlet; the first onward flow channel continuing successively to and in fluid communication with a microfluidic DEP device as herein described.

The two onward flow channels may comprise parallel flow channels and for example respective upper and lower parallel flow channels.

ACEO is a known technique for flow control. A microfluidic ACE° device for use in embodiments of the invention following these principles comprises a microfluidic reactor cell defined in the microfluidic pathway through the concentrator module provided with first and second electrodes which are spaced either side of the reactor cell such as to generate in use a suitably controlled AC electric field across the reactor cell to induce the desired flow control effect.

DEP is a known technique in microfluidics for particle or cell separation. It exploits the phenomenon that dielectric particles in a non-uniform electric field are subject to a force that can be used to separate and concentrate them. DEP is generated by small interdigitated electrodes operating at high frequencies. Positive DEP attracts particles towards the electrode edges. Negative DEP pushes particles away from the electrodes. DEP can be used therefore to trap and immobilise particles at the DEP electrodes.

A microfluidic DEP device for use in embodiments of the invention following these principles comprises a microfluidic reactor cell defined in the microfluidic pathway through the concentrator module provided with first and second electrodes which are spaced either side of the reactor cell such as to generate in use the required nonuniform electric field across the reactor cell.

Microfluidic DEP devices have been considered for biological sample concentration but DEP is conventionally targeted at a specific target. Applicant's desired solution is to investigate multiple targets and in particular to target bacterial and viral DNA and viral RNA. The combined approach is uniquely suited to this.

At its simplest, the two stages of the basic embodiment of the first aspect of the invention will produce a final output of the release phase in which larger particles such as bacteria are separated and concentrated and a first output of the capture phase, comprising the rest of the sample and potentially including further useful material.

This may simply be discharged, but more preferably in a dual system is subject to processing in a further microfluidic processing unit that is operable to preferentially divert a further target biological material to a second output outlet. For example, the second target material is viral material.

The further microfluidic processing unit may include a microfluidic DEP device and/ or a microfluidic ACED device in appropriate configuration and is for example a microfluidic ACED device of the second aspect of the invention.

Particularly advantageously as DEP can size select, the dual system can send streams to different pipelines. This may for example allow separation of bacterial and viral material when processed via subsequent modules.

Advantageously therefore, one or more microfluidic pathways may be configured to receive and process a fluid stream comprising a biological sample including bacterial and viral material in a buffer fluid and to produce an output flow in which a relative concentration of the bacterial material is increased, and optionally further a discharge flow in which a relative concentration of other material is increased.

Additionally or alternatively advantageously therefore, one or more microfluidic pathways may be configured to receive and process a fluid stream comprising a biological sample including bacterial and viral material in a buffer fluid and to produce an output flow in which a relative concentration of the viral material is increased, and optionally further a discharge flow in which a relative concentration of other material is increased.

Advantageously therefore, in some embodiments, the concentrator module is a dual system that includes a first set of output outlets thereby adapted preferentially to output concentrated bacterial material, a second set of output outlets thereby adapted preferentially to output concentrated viral material, and a set of discharge outlets to output material/ fluid to be discarded.

A concentrator module in accordance with the first aspect of the invention may optionally further include additional microfluidic processing units fluidly in series with the principal microfluidic ACEO device and principal microfluidic DEP device of the basic embodiments, and operable to complement or enhance the concentration function.

For example in some embodiments, a physical filter may be disposed upstream of the principal microfluidic ACED device and principal microfluidic DEP device.

For example in some embodiments, a secondary microfluidic DEP device may be provided upstream of the principal microfluidic ACEO device and principal microfluidic DEP device and configured to be operable to generate a negative DEP such as to tend to urge material towards a region of vortex generation of the principal microfluidic ACEO device. This may allow larger initial flow channels.

Thus, the invention of the first aspect provides microfluidic concentrator module solutions based on ACED and DEP principles in which the concentrator module is effective for targeting for concentration cellular material such as bacterial material from a metagenomic sample, and which may also be modified for the separate targeting for concentration concentration of viral material.

In accordance with the invention in a second aspect, a concentrator module to receive a sample comprising biological material in a fluid buffer medium and increase the concentration of a target biological material present within the sample comprises a microfluidic apparatus including: an inlet, an output outlet, at least one further outlet, and a fluid pathway therebetween including a processing unit that is operable to preferentially divert the target biological material to the output outlet, and for example preferentially diverts buffer medium/ undesired solid material/ non-target biological material to the further outlet, for example to be discarded or further processed; wherein the processing unit includes a microfluidic ACE° device comprising one or more microfluidic channels defining an initial flow direction through the device and an ACEO electrode system disposed at an angle to the flow direction.

By analogy, a method to process a sample comprising biological material in a fluid buffer medium and increase the concentration of a target biological material present within the sample comprises passing the biological material and fluid buffer medium through a concentrator module in accordance with the second aspect to increase the concentration of the biological material.

Applicant's concentrator module of the first aspect of the invention relies upon parallel ACE° and DEP in series to target larger components of the biological material, for example over 1 micron in size, and for example bacteria, to tend to preferentially segregate and concentrate such material by capturing at the DEP electrodes and separating into a first process flow stream to be diverted to an output outlet. The DEP electrodes are not effective at capturing smaller components of the biological material, for example below 1 micron in size, which may include viral particles.

By contrast, applicant has found that a microfluidic ACE° device modified such that the ACE° electrode system disposed at an angle to the flow direction defined by the microfluidic channels therein may be surprisingly effective at tending to preferentially segregate and concentrate such material, and thus may be effective at separating viral particles from a process flow. Thus, in accordance with the invention of the second aspect, a typical target biological material comprises viral material.

Suitably, the microfluidic ACE° device of this aspect of the invention comprises one or more inlet microfluidic channels defining an initial flow direction through the device and an ACED electrode system disposed at an angle to the flow direction electrode system comprising a plurality of parallel electrodes at an angle to the initial flow direction, which may thereby created when operated a plurality of outlet flows into which smaller particles, for example below 1 micron in size, tend to be concentrated.

A suitable angle may be 2 to 35 degrees to a direction parallel to the initial flow direction through the device.

In some embodiments, a microfluidic DEP device may be provided upstream of the microfluidic ACEO device configured to be operable to generate a negative DEP such as to tend to urge material towards a region of vortex generation of the microfluidic ACE° device. This may allow larger initial flow channels.

Thus, the invention of the second aspect provides microfluidic concentrator module solutions based on modified ACED and optionally further DEP principles in which the concentrator module is effective for targeting for concentration smaller particles of material such as viral material from a metagenomic sample.

The invention of the second aspect may be particularly suited to application to a sample comprising biological material in a fluid buffer medium from which bacterial material has already been removed in accordance with the module or method of the first aspect of the invention.

Thus, a possible dual system operable to concentrate separately for separate processing both bacterial material and viral material from a metagenomic sample may comprise a concentrator module embodying the principles of both the first aspect and the second aspect, in particular being disposed to act sequentially and for example being fluidly in series.

That is to say, for example, a dual system in a third more complete aspect of the invention comprises: a concentrator module to receive a sample comprising biological material in a fluid buffer medium and separate and increase the concentration of a first and a second target biological material present within the sample comprises a microfluidic apparatus including fluidly in series: an inlet; an inlet flow channel in fluid communication with a first microfluidic ACED device comprising one or more microfluidic channels defining an initial flow direction through the device and an ACEO electrode system disposed in parallel to the flow direction, and operable to tend to cause biological material to segregate into a first part of the flow channel; at least first part of the flow channel being fluidly continuous with an onward flow channel continuing successively to and in fluid communication with a microfluidic DEP device and a second microfluidic ACED device comprising one or more microfluidic channels defining an initial flow direction through the device and an ACE° electrode system disposed at an angle to the flow direction; selectively closable first and second separation channels into which the onward flow channel divides fluidly downstream of the microfluidic DEP device; the first separation channel continuing to a first target material outlet; the second separation channel continuing to a second target material outlet.

Optionally, the inlet flow channel is divided downstream of the first microfluidic ACE° device into a first onward flow channel dividing from the same in such manner as to be fluidly continuous with the first part of the flow channel, and a second onward flow channel; the second onward flow channel optionally continuing to a discharge outlet; the first onward flow channel continuing successively to and in fluid communication with a microfluidic DEP device and a second microfluidic ACEO device as herein described.

By analogy, a method to process a sample comprising biological material in a fluid buffer medium and increase the concentration of a target biological material present within the sample comprises passing the biological material and fluid buffer medium through a concentrator module in accordance with the second aspect to separate first and second target materials and to increase the concentration of the biological material provided to separate first and second target material outlets.

In operation, the system in the third more complete aspect of the invention comprises features and advantages of the first and second aspects in a manner which will be understood.

For example, two stages of operation are employed, comprising: a first phase in which: the first ACED electrode system is operated to tend to cause biological material within the sample to be urged towards the electrode system; optionally, material so urged is transmitted through a first onward flow channel and the reminder of the flow is divided to the second onward flow channel; the DEP electrode system is operated with positive DEP to trap the first target from the flow drawn into its vicinity by the ACE° electrode system, for example being in the first onward flow channel; material not so captured flows through and beyond to the second ACE° electrode system; the second ACED electrode is operated to urge the second target material into the second separation channel and through the second target material outlet; a second phase in which: the second separation channel is closed; the DEP electrode system is not operated or is operated with negative DEP to release the trapped larger particles and cause the same to pass into the first separation channel and through the first target material outlet.

In particular preferably, the first target material may for example be bacterial material and the second target material may be viral material. Thus, the invention of the third aspect provides microfluidic concentrator module solutions based on conventional and modified ACE° and DEP principles in which the concentrator module is effective for targeting for separation and concentration both bacterial and viral material from a metagenomic sample Preferred and advantageous embodiments of the third more complete aspect of the invention will be understood by analogy with the description of the first and second aspects.

For the reasons discussed herein, the particular features of embodiments of collector module according to the principles of the invention produces synergistic advantages that go beyond the mere routine advantages of microfluidic systems.

In particular, the potential to exploit and for example combine DEP and ACE° principles in accordance with the principles described herein gives an ability to develop a microfluidic concentration module that might be adapted for use in conjunction with a system and method for the concentration of non-specific metagenomic biological samples that is susceptible of automation/ use in the field/ that can process for subsequent preparation for analysing a sample comprising a range of bacterial and viral material from multiple targets to a degree of functionality not hitherto possible even in know lab-on-chip methodologies.

In particular, the ability to provide a system and method that can automatically separate viral and bacterial material using the size selection capability of DEP, in embodiments enhanced by the use of ACE(); and thereby to send viral and bacterial streams to different pipelines for further and different processing, is a powerful tool in achieving this further objective of developing a system and method for the concentration of non-specific biological samples including both viral and bacterial material (such as might be expected in an environmental sample) for further processing for analysing in a single integrated and automatable system.

As discussed, the more complete aspect of the invention is in particular concerned with the processing and preparation for analysis of environmentally collected biological material samples that can be expected to contain a wide range of different biological materials and the extraction of genetic material therefrom for analysing. It is particularly desirable if this could be done in-line in an automated and at least semi-continuous manner with a sample collector, for example out in the environment under investigation. The presence of a wide range of different biological materials means the presence of a wide range of genetic material and in particular the presence of DNA and RNA from multiple sources which creates particular difficulties.

Each of the stages of the process herein described is known generally in the field of extraction of DNA/ RNA material for analysing, particularly where this is carried out as a batch process and on a laboratory scale. The development of microfluidic reactors and processors for certain stages of the process have been proposed as having advantages for example in relation to miniaturisation or automation.

However, the often conflicting demands of the various preparation stages that are required to move from an environmentally collected sample with a wide range of varied biological material present to the targeted analysis to detect the presence of particular specific target sequences identifying specific target biological hazards or pathogens has proved resistant to automation and miniaturisation techniques.

Applicant has successfully overcome these potentially conflicting demands to produce a system that offers the potential for such processing of samples containing a wide range of genetic material and in particular both DNA and RNA from multiple sources in-line with the collector and in at least a partly automated manner and potentially in real time.

Applicant has arrived at a solution in part through the provision of a system that processes together both DNA and RNA from multiple sources for subsequent multiplex analysis rather trying to separate specific target fragments, and in part by the particular features of the collection module described herein and each other module discussed in further detail below.

In accordance with this approach, each module is conformed as a microfluidic system, and the successive modules may in preferred embodiments then be successively arrayed in compact manner to provide a complete solution based partially or fully on lab-on-chip principles with the potential for partially or fully automated throughput, for example in-line with an upstream collection system and/ or a downstream genomic sequencing system.

In general terms, microfluidics is an established concept and the skilled person would entirely appreciate the difference between the microfluidic conformance of the modules comprising applicant's invention and larger scale laboratory batch process modules. Microfluidic systems include as will be familiar systems that manipulate and transport, mix, separate, or otherwise process low volumes of fluids on a small (typically sub millimetre) scale. Each module of the system of the invention is conformed as a microfluidic system in that at least some of its fluid manipulation elements are conformed on a microfluidic scale, and in particular in that inlet and outlet fluid pathways are on a microfluidic scale. As will be understood from discussion of the preferred embodiments, other elements of a module may embody principles not conventionally seen as microfluidic to provide a complete solution a complete solution based partially or fully on lab-on-chip principles.

Each module comprises one or more fluid inlets, one or more fluid outlets, and one or more fluidly continuous pathways between the inlets and the outlets that act to transfer fluid between the inlet(s) and the outlet(s) via suitable fluid processing units.

Unless the context clearly demands otherwise, reference herein to an inlet encompass a plurality of inlets, references herein to an outlet encompass a plurality of outlets, references herein to a fluid pathway include a plurality of fluid pathways each defining a parallel fluid communication between a respective one or more inlets and a respective one or more outlets. Reference to first, second etc inlets, outlets and pathways with differing functions encompass first, second etc sets of inlets, outlets and pathways with the respective differing functions. Each module comprises a microfluidic module and accordingly each inlet, outlet, pathway and processing unit is preferably respectively a microfluidic inlet, outlet, pathway or processing unit as the case may be. Unless the context clearly demands otherwise, reference herein to a processing volume or chamber or the like of a processing unit does not imply any particular structure but indicates a zone of the fluidly continuous pathway in which a fluid process takes place.

Advantageously thereby, in each module one or more microfluidic pathways may be configured to receive and process a fluid stream comprising a biological sample from a preceding process module as herein described, to pass the same through a microfluidic processing module, and to produce an output flow of a suitable modified fluid stream to be supplied to a succeeding process module as herein described.

A module may optionally be configured to produce plural different output flows of a differently modified fluid stream to be supplied to a succeeding process module as herein described. That is, a module may be configured to comprise a first set of output flows and second set of output flows. A module may optionally further be configured to produce a discharge flow to be discharged.

In some embodiments of the system, a module may comprise plural sets of pathways, each respective pathway of a set of pathways configured to pass fluid through a microfluidic processing module configured for a particular process to provide a respective set of output flows. In such embodiments different sets of pathways may thereby be configured to perform different processes.

In such embodiments, each set of pathways may be served by a fluidly separate set of inlets or serve a fluidly separate set of outlets or both. Alternatively plural sets of pathways may share a common set of inlets. Further alternatively plural sets of pathways may share a common set of outlets.

In embodiments of the system where a preceding module is configured to produce plural sets of output flows, the output flows of each respective set are conveniently provided in fluid communication with a respectively discrete set of inlets of a succeeding process module.

An example microfluidic module system that might be adaptable to the principles of the present invention is described in PCT/GB2022/050889. The disclosure described, and embodiments of the present invention may include or make use of: a microfluidic system comprising a plurality of fluidly connected microfluidic chambers, each microfluidic chamber comprising: a fluid sample inlet; a fluid sample outlet; a selectably closable valve operable to enable gas to be vented from the chamber; a pressurisation system operable to apply an overpressure to one or more first microfluidic chambers being fluidly most upstream; or a microfluidic method comprising providing a microfluidic system according to any preceding claim; supplying a fluid sample to the one or more first microfluidic chambers being fluidly most upstream; operating the pressurisation system to apply an overpressure to the one or more first microfluidic chambers; selectively operating the valves of the fluidly connected microfluidic chambers to cause the fluid sample to move successively between the microfluidic chambers.

Each microfluidic chamber may comprise a microfluidic reactor defining a processing volume and having a processing function, at least one of which comprises a concentrator module according to the first aspect hereinabove described. The microfluidic chambers are disposed fluidly successively to enable performance of their functions successively. Each microfluidic chamber comprises a valved outlet configured to be operable to equalise pressure within the chamber when the valve is in an open position, but is otherwise sealed to the ambient environment of the system. A microfluidic chamber may include additional inlets/ outlets for reagents and outputs associated with such a function, but these are again so arranged that the chamber is closed to the ambient environment of the system (where ambient environment herein means the environment immediately external to the system, whether that is ambient atmosphere or a closed and for example inert gaseous system).

The system thus comprises a series of individual sealed microfluidic reactors, connected together to form a successive array. Such is familiar. The invention is characterised by the provision of a pressurisation system to apply an overpressure to the first chamber and by the provision in each microfluidic chamber of a valve operable to enables gas to be vented from the microfluidic chamber, and thus operable when open to equalise pressure therein to that of the ambient environment of the system.

In use, the pressurisation system applies an overpressure, preferably a constant overpressure, to at least a first one of the chambers being fluidly most upstream.

This creates a constant pressure differential between the fluidly upstream and the fluidly downstream end of the series of chambers. It is then possible, merely by selective opening and closing of the valves, for the fluid sample to be transferred from one chamber to the next, and for example thereby from one process to the next.

This transportation successively through the series of chambers is achieved simply by operation of the valves appropriately sequentially, and by action of the pressurisation. It can be seen that if a chamber has its valve open any overpressure at the inlet is equalised by venting of gas from the chamber, the fluid remains in the chamber and the fluid can be acted upon by the processes of the reactor volume created therein. Once the valve is closed, the build up of pressure in the chamber forces fluid through the outlet of the chamber to an inlet of a subsequent chamber in fluid series thereto, to which the outlet of the preceding chamber is fluidly connected.

Thus, a series of microfluidic chambers comprising in the typical case a series of microfluidic reactor volumes can be used to create a microfluidic chain of a series of processes acting upon the fluid.

References herein to an output flow will be understood to suggest flow for use in a subsequent stage in the process for example being directed through an outlet comprising an output outlet in fluid communication with an inlet of a subsequent module as described herein. References herein to a discharge flow will be understood to suggest flow to be discarded and not passed to a subsequent stage in the process for example being directed through an outlet comprising a discharge outlet.

The invention is adapted for use with a biological sample collected into a fluid buffer and for example an aqueous buffer. After collection the sample is processed by the system of the invention which is conformed as an automatable microfluidic wet-lab.

The wet lab process is made up of a number of process steps performed by respective modules/ as respective process steps.

Preferred and characterising features of each module of the system of the first aspect of the invention, and the way in which these interact to give the possibility of an integrated and compact lab-on-chip solution with the potential for use in-line in real time with an upstream collection system, are discussed below. Preferred and characterising features of the steps of the method of the second aspect of the invention will be appreciated by analogy.

The concentrator module/ concentration step herein described is adapted to take an input flow of a relatively large volume of collected biological material/ fluid buffer and reduced into a much smaller volume of liquid to enable it to be processed by the rest of the system. The concentrator module acts such that biological material, and in particular biological material from which DNA/ RNA may be extracted, is retained, but both some buffer fluid and undesired solid material including undesired organic material is removed.

The concentrator module / concentration step produces an output flow in which the material from which DNA/ RNA may be extracted is substantially concentrated. In preferred embodiments above described, the concentrator module / concentration step may be adapted to produce a dual flow including a first output flow in which bacterial material is particularly concentrated and a second output flow in which viral material is particularly concentrated. In other embodiments a common output flow may include concentrations of bacterial and viral material from which DNA/ RNA may be extracted.

An advantage of preferred configurations of the concentrator module / concentration step is the ability to produce a dual flow including a first output flow in which bacterial material is particularly concentrated and a second output flow in which viral material is particularly concentrated.

As discussed, as techniques for analysis become more rapid, and more susceptible of miniaturisation, systems that provide for at least some in line analysis steps to be performed on a collected sample are attractive. It is a particular object of the module and method of the invention that they are susceptible to such use in-line in real time with an upstream collection system and/ or a downstream further processing system.

Embodiments of the invention further concern the collection of a sample from the environment, and for example from the air at a monitored location or site, into an aqueous buffer and the processing and preparation of biological samples from the collected sample, in particular in-line with the sampling stage. The sampling stage may make use of such a sampler as described UK patent application No 2003303.1, although the invention is not limited to this. The invention finds particular applicability to the collection and processing and preparation of and optionally further the analysis of biological samples collected by such a sampler, and for example the isolation of genetic material and optionally further the analysis of genetic material.

Desirably the invention in a more complete aspect provides a collection and processing system comprising: a collection system to collect a sample of environmental biological material, for example comprising aerosolised biological particulate material, into a fluid buffer; a module for concentration or a more complete system for processing and preparation for analysis as above described in fluid communication with the same, and thereby configured as the case may be to concentration or process and prepare for analysis biological material collected by the collection system in use.

UK patent application No 2003303.1 describes an air sampler that collects airborne particulates into a liquid collection buffer. In preferred embodiments, the system for processing and preparation for analysis of the first aspect of the invention is adapted for use with such an air sampler. In In preferred embodiments, the collection and processing system of the further aspect of the invention comprises such an air sampler.

The air sampler described in UK patent application No 2003303.1 comprises in general terms: a sampler inlet to receive a gas flow comprising aerosolised particulate material; a prefilter module comprising a first inertial classifier fluidly positioned to receive an inlet flow from the collector inlet and configured to divide the inlet flow into a first outlet flow into which particles smaller than a predetermined cut point size tend to segregate differentially and a second outlet flow into which particles larger than a predetermined cut point size tend to segregate differentially, the predetermined cut point size being selected to be at a desired maximum particle size; a concentrator module comprising a second inertial classifier fluidly positioned to receive an inlet flow from the first outlet flow of the first inertial classifier and configured to divide the inlet flow into a first outlet flow into which particles smaller than a predetermined cut point size tend to segregate differentially and a second outlet flow into which particles larger than a predetermined cut point size tend to segregate differentially, the predetermined cut point size being selected to be at or below a desired minimum particle size; optionally at least one further inertial classifier fluidly positioned to receive an inlet flow from the second outlet flow of the second inertial classifier and configured to divide the inlet flow into a first outlet flow into which particles smaller than a predetermined cut point size tend to segregate differentially and a second outlet flow into which particles larger than a predetermined cut point size tend to segregate differentially, the predetermined cut point size being selected to be at or below a desired minimum particle size; and an outlet to output the second outlet flow of the concentrator module; a collection module positioned fluidly to receive the output of the air concentrator module and capture the particles into an aqueous liquid buffer.

The air sampler collects large amounts of air (and therefore biological material). It is distinctly characterised by the arrangement of multiple inertial classifiers fluidly in series to perform both a large particle filtration function and a sample concentration function, and the further provision of a collection module to collect the sample into an aqueous liquid buffer. In use with the system of the present invention the sample in aqueous liquid buffer is supplied to the concentration module, and the concentration module is accordingly positioned fluidly downstream of the air sampler.

The first function is performed by the pre-filter module comprising a first inertial classifier, which is disposed such that the large particle outlet flow is discarded and the small particle outlet flow is retained for further processing.

The concentrator module has a further inertial classifier stage with at least a second inertial classifier and optionally at least one further inertial classifier stages which are disposed with the opposite arrangement. With appropriate selection of cut size these at further stage(s) can be used to concentrate the particles into a smaller volume.

Suitable inertial classifiers that produce the desired first outlet flow into which smaller particles tend to segregate differentially and a second outlet flow into which larger particles tend to segregate include virtual impactors and cyclones.

Virtual impactors are particularly preferred. In preferred embodiments of the sampler, each of the inertial classifiers comprises a virtual impactor. That is, in such embodiments, the sampler system for the collection of aerosolised particulate material comprises: a sampler inlet to receive a gas flow comprising aerosolised particulate material; a prefilter module comprising a first virtual impactor fluidly positioned to receive an inlet flow from the collector inlet and configured to divide the inlet flow into a major outlet flow into which smaller particles tend to segregate differentially and a minor outlet flow into which larger particles tend to segregate differentially; a concentrator module comprising a second virtual impactor fluidly positioned to receive an inlet flow from the major outlet flow of the first virtual impactor; optionally at least one further virtual impactor fluidly positioned to receive an inlet flow from the minor outlet flow of the second virtual impactor; and an outlet to output the minor outlet flow of the concentrator module; a collection module positioned fluidly to receive the output of the air concentrator module and capture the particles into an aqueous liquid buffer.

More completely, in such embodiments, the system for the collection of aerosolised particulate material includes: a sampler inlet to receive a gas flow comprising aerosolised particulate material; a prefilter module comprising a first virtual impactor fluidly positioned to receive an inlet flow from the collector inlet and configured to divide the inlet flow into a major outlet flow into which smaller particles tend to segregate differentially and a minor outlet flow into which larger particles tend to segregate differentially; a concentrator module comprising a second virtual impactor fluidly positioned to receive an inlet flow from the major outlet flow of the first virtual impactor and configured to divide the inlet flow into a major outlet flow into which smaller particles tend to segregate differentially and a minor outlet flow into which larger particles tend to segregate differentially; optionally at least one further virtual impactor fluidly positioned to receive an inlet flow from the minor outlet flow of the second virtual impactor and configured to divide the inlet flow into a major outlet flow into which smaller particles tend to segregate differentially and a minor outlet flow into which larger particles tend to segregate differentially; and an outlet to output the minor outlet flow of the concentrator module; a collection module positioned fluidly to receive the output of the air concentrator module and capture the particles into an aqueous liquid buffer.

The key to the sampler is the particular arrangement of major and minor flow whereby the respective virtual impactors are used to perform both a large particle filtration function and a target particle concentration function.

An optional physical prefilter may additionally be used upstream of the prefilter module, for example as part of the gas sampler inlet, to remove even larger particles from the system altogether before they are drawn into the first virtual impactor. A suitable filter might include a wire mesh for example.

Suitable impeller means may be provided to draw air through the sampler inlet and into the prefilter module.

In some embodiments a virtual impactor has a generally circular cross section. In some embodiments this may taper in an outlet direction. For example the virtual impactor may comprise cylindrical body portion and a tapered and for example conical outlet portion.

Preferably, the virtual impactor has a generally circular inlet and a generally circular outlet. Preferably, successive virtual impactors are directly linked to form a fluidly continuous structure. For example successive virtual impactors may be mounted in line linked with circular ducts to form a fluidly continuous structure.

Successive virtual impactors may be sized to reflect the differential flow volumes, a downstream virtual impactor being smaller than an upstream one.

In some embodiments the virtual impactor defines a plurality of flow channels. In some embodiments the virtual impactor defines a plurality of concentric arcuate flow channels. Such an arrangement may be advantageous in allowing successive virtual impactors to be linked by circular inlet/ outlet ducts.

In some embodiments respective outlet channel portions of the plurality of flow channels are differentially structured and for example differentially tapered to create a more even distribution of flow. For example in the case where the virtual impactor defines a plurality of concentric arcuate flow channels these may be larger towards the outside.

Conveniently, the collection module comprises a wet cyclone.

Other possible features of an air collector for use with the system for processing and preparation for analysis of the first aspect of the invention to constitute the collection and processing system of the further aspect of the invention may be appreciated from UK patent application No 2003303.1.

In preferred embodiments, the collection and processing system of the further aspect of the invention is adapted to be used in the field using portable apparatus, and in particular using vehicle mounted apparatus.

For example the system components are compactly associated with together in portable manner, and for example adapted to be mounted on a vehicle.

In further embodiments of the invention, a vehicle comprising such an apparatus is provided.

In other preferred embodiments, the collection and processing system of the further aspect of the invention is adapted to be performed on an enclosed space using apparatus deployed in that space. This may be particular applicable for a space that has a partly recirculated atmosphere, and may be included in line with the recirculation system. For example the apparatus is deployed in an enclosed room, vehicle or aircraft to detect communicable airborne pathogens. Particularly advantageously in an aircraft this may be able to produce results over the course of a single journey alert of a threat before the plane lands.

The concept of the invention in an extended aspect comprises the use of a system or method as herein before described as part of a system or a method for the analysis of aerosolised particulate material.

In such an extended aspect, the system may comprise further modules, and the method may comprise further steps, in relation to such an analysis. In particular the system may comprise further modules, and the method may comprise further steps, for isolation of genetic material and its analysis.

Brief Description of Drawings

The invention will now be described by way of example with reference to the accompanying drawings in which: Figures 1 and 2 respectively show a schematic top view, and a side view in section, of a combined ACED and DEP system intended to achieve concentration of larger particles on the scale of bacterial cells, and thus providing an example embodiment of a first aspect of the invention; Figure 3 illustrates an adaptation of an ACEO system intended to achieve concentration of smaller particles on the scale of viral particles, and thus providing an example embodiment of a second aspect of the invention; Figures 4 and 5 show the two principles embodied in figures 1 to 3 combined in a complex capture and concentration system illustrative of an embodiment of a more complete aspect of the invention; Figure 6 shows a simple schematic of a linear chain of microfluidic reactors applying an overpressure principle with fluid flow left to right.

Detailed Description

The concept of the invention in its broadest aspect is a system and method for the concentration and/ or separation of biological samples, and in particular metagenomic samples. In particular, the invention concerns the concentration and/ or separation of bacterial and viral materials from metagenomic biological samples.

The concept in a more extended aspect provides for subsequent processing and preparation, for analysis of the samples, and in particular for molecular biological analysis. In preferred embodiments, the invention concerns the analysis of environmentally collected biological samples, for example of airborne biological material which has been collected into a suitable aqueous buffer.

The concept of the invention in a further extended aspect comprises the use of an air sampling and collection system and method as part of a system and method for the preparation and analysis of aerosolised particulate biological material, for example making use of an air sampling and collection system and method such as described in UK patent application No 2003303.1.

The invention will now be described in this context with reference to the example embodiment shown in the drawings and described in detail below.

In some scenarios it may be desirable to concentrate and separate larger components of a metagenomic sample, and in particular bacterial components. Figures 1 and 2 respectively show a schematic top view, and a side view in section, of a combined ACEO and DEP system suitable for effecting concentration of larger biological particles, and in particular of cellular material such as bacterial material, for subsequent processing, and thus providing an example embodiment of a first aspect of the invention.

In the illustrated embodiment, an alternating current electroosmosis (ACEO) electrode system and a dielectrophoresis (DEP) electrode system are positioned fluidly consecutively in series in a microfluidic channel. The ACED electrode system is disposed parallel to a flow direction in the microfluidic channel, and in the illustrated embodiment is at the bottom of the channel. The DEP electrode system is disposed parallel to a flow direction in the microfluidic channel and positioned immediately fluidly downstream of the ACEO electrode system and located in the same relative position, in the illustrated embodiment at the bottom of the channel. Other arrangements of plural parallel electrodes consistent with the principles of operation described below could be imagined.

Two phases of operation are respectively illustrated in figures 1 and 2, with figure 1 showing a capture operation in which larger organic particles such as cellular materials are captured at the DEP electrodes and the aqueous buffer is discarded, and in figure 2 the trapped particles are then released. The embodiment is in particular concerned with trapping, separating and concentrating bacteria from a metagenomic sample for subsequent processing.

The physics behind both ACE° and DEP is generally known.

In ACEO, electrodes powered at low frequencies generated vortices that are used to pull particles from the bulk liquid and push them down towards the electrodes. The ACE° effect is generally independent of particle size, and can be expected to tend to cause all biological materials within the sample, including bacteria, viruses, proteins, and various fragments of the same, to be pushed urged towards the electrodes, and in the case of the embodiment illustrated to be urged down in the channel towards the electrode system at the bottom.

DEP is generated by small interdigitated electrodes operating at high frequencies. Positive DEP attracts particles towards the electrode edges. Negative DEP pushes particles away from the electrodes. DEP can be used therefore to trap and immobilise particles at the DEP electrodes. However, positive DEP trapping forces decrease with electrode width and distance from the electrode surface. Trapping force is increased with particle size. As a result, with the geometries typical for microfluidic systems, trapping forces are likely to be negligible at particle sizes below about 1 micron, and consequently negligible for viral particles.

Thus, the combination in series of a downstream DEP electrode system parallel to the flow in the microfluidic channel which is capable of trapping larger particles such as bacteria and an upstream ACE° system similarly aligned parallel to the flow in the microfluidic channel which tends to pull all particles, including larger particles such as bacteria towards the surface where the parallel DEP electrode is disposed, allowing it to act effectively, creates in combination the opportunity preferentially to capture and therefore to concentrate larger particles from a meta-genomic biological sample, and in particular to capture and concentrate bacteria preferentially.

The exploitation of this principle in the embodiment of figures 1 and 2 is shown. In a simple example design two electrodes are provided with a straight channel. The first electrode generates ACE° and pulls the organic particles, and in particular the bacteria within the sample, from the bulk liquid into a concentrated stream or streams parallel to the electrodes. In the case of the in the case of the embodiment illustrated they are pulled down in the channel towards the electrode system at the bottom.

The flow channel will be suitably sized with respect to the electrode arrangement applying familiar principles. While larger channels might generate higher potential flow rates, the requirement for the vortex effect to be exploited is likely to impose limits in practice to the extent of the microfluidic channel in the vicinity of the electrode system. In the simple parallel electrode system of the embodiment a channel depth of 200-250 pm might be appropriate.

Pulling the bacteria using the ACE° effect brings them into reach of the DEP effect generated by the second electrode system, which is strongest closer to the electrode surface. The second electrode system is used to generate positive DEP forces that will trap the bacteria while the rest of the sample passes through. The DEP effect is generated by small interdigitated electrodes operating at high frequencies Principles of DEP electrode design will be familiar and the skilled person will apply them as appropriate. In the example illustrated system individual interdigitated DEP electrodes are envisaged with 16 pm width 46 pm spacing.

In the embodiment the channel is divided immediately downstream of the ACED electrode system into a bottom channel, for example 50 pm deep, with the DEP electrode system at the bottom, and a top channel, for example 100 pm deep. This simple arrangement is illustrative only and complex arrangements of multiple channels could be envisaged consistent with the principles of operation described.

The biological material is drawn into the lower part of the main channel by the ACED system and preferentially segregated into the bottom channel while much of the fluid is passed through the top channel where it may simply be discharged.

The trapping phase is illustrated in figure 1. The bacteria are trapped in the bottom channel at the positive DEP electrodes as shown. Other smaller biological material, which may for example include viruses, is generally too small to be substantially affected by the DEP forces and is not so trapped.

In a release phase, the top channel is then blocked and a negative DEP applied to the second electrode system releasing the trapped bacteria which can then pass along the bottom channel for further processing.

At its simplest, the embodiment of figures 1 and 2 may be used to concentrate larger particles which are more susceptible to the action of the DEP electrodes, and in particular may be used to concentrate bacteria from a metagenomic sample in aqueous buffer, which has for example been collected environmentally making use of the air sampling and collection system above referenced. Most of the aqueous buffer can be discharged, so that the bacterial content of the sample is significantly concentrated for further processing.

The bulk fluid, and other biological material not so concentrated at the DEP electrode system, which may for example include viruses which are generally too small to be substantially affected by the DEP forces, passed through the system during the concentration phase illustrated in figure 1. They may simply be discarded with the bulk fluid. Alternatively, further processing steps may be built in to isolate further biological material samples from this discharge stream.

In other scenarios, either additionally or alternatively to the above, it may be desirable to concentrate and separate smaller components of a metagenomic sample, and in particular viral components. Figure 3 illustrates an adaptation of an ACE° system intended to achieve concentration of smaller particles, for example below 1 micron in size, and thus providing an example embodiment of a second aspect of the invention, and in particular being intended to target viruses from the sample.

At its most general, it thus represents an embodiment of a concentrator in accordance with the second aspect of the invention described above, and in combination with the concentrator of figures 1 and 2 offers an improved combined system capable of separating and concentrating bacterial and viral particles, as presented in the example embodiment of the third aspect of the invention shown in figures 4 and 5.

This embodiment follows from the recognition that DEP cannot be used to concentrate viral particles, because the forces are too small for nanometre sized particles. This has led in consequence to the realisation that only ACE° can be used. However this requires a modification of conventional ACE° principles in order to effect the required concentration. In contrast to the conventional electrode system used in figures 1 and 2, in which the ACE° electrodes are parallel to the flow in the microfluidic channel, the illustrated embodiment uses large electrodes angled to the direction of flow to focus the particles into a single concentrated stream to be collected on a side channel.

The two principles embodied in figures 1 to 3 are combined in the complex capture and concentration system proposed in figures 4 and 5, which respectively show operation during a viral concentration step and during a bacterial release step. As previously, an environmentally collected metagenomic sample in fluid and for example aqueous buffer is passed into the microfluidic system from the left.

In this embodiment, an additional first DEP electrode system is provided fluidly upstream of the ACEO electrode system. The bacteria and viruses are drawn inside the device and pushed away from the top of the channel using the negative DEP effect of this first electrode patterned at the top of the channel. This refinement allows for the channel heights to be increased beyond the ACE° vortex height, allowing for higher initial throughput.

The first DEP electrode brings the particles within the range of the maximum height of the vortex generated by the ACED electrodes patterned on the bottom of the channel and the particles are then pushed down towards the bottom of the channel where they are diverted at a point where a top channel and bottom channel divide.

The particles are preferentially segregated into the bottom channel whilst the bulk fluid is preferentially segregated into the top channel, for example for discard to waste.

The bacteria are then trapped by the parallel DEP electrodes in the bottom channel in accordance with the principles described with reference to figure 1. The viruses are not influenced by the DEP electrodes but are concentrated further downstream using slanted ACED electrodes operating on the principles of figure 3. At the step represented in figure 4 this tends to produce a concentration of viral particles through the lowermost outlet.

Once the sample is passed through, as shown in figure 5, the top discharge outlet and the lowermost viral outlet are closed, and the DEP electrodes are turned off/reversed to release the bacteria which can then be collected through the third, middle outlet.

Thus, the arrangement represented in figure 4 and 5 has to advantageous functional effects. It concentrates the biological material in the sample by retaining target bacterial and viral materials while discharging the bulk of the fluid, and it effectively separates a stream in which viral particles are preferentially segregated to predominate and a stream in which bacteria are preferentially segregated to predominate, so that each such stream can be separately processed as required.

An example microfluidic module system that might be adaptable to the principles of the present invention is described in PCT/GB2022/050889. That describes a plurality of fluidly connected microfluidic chambers arranged with a pressurisation system operable to apply an overpressure to one or more first microfluidic chambers being fluidly most upstream. The microfluidic chambers are disposed fluidly successively to enable performance of their functions successively. The principles are illustrated in figure 6.

The basic schematic of figure 6 shows a simple serial arrangement of individual sealed micro-bioreactors, microfluidically connected together as shown and each with a valve outlet through which, with the valve open, the chamber may be vented (not shown in figure 1). Pressure means (not shown in figure 1) create a pressure differential with a higher pressure to the left. By having a constant pressure differential at one end of the chain to the other, by opening and closing valves the fluid can be transferred from one bioreactor to the next.

At the first instance, if a bioreactor has its valve open to the ambient the pressure source can force the liquid into the bioreactor via its inlet. With the valve open, the fluid remains in the bioreactor and the fluid can be acted upon by the processes of the bioreactor. Once the valve is closed, the pressure builds up in the bioreactor and the fluid is forced to the outlet of the bioreactor towards the next bioreactor in the chain. Thus a series of bioreactors can be used to create a microfluidics chain of a series of processes acting upon the fluid.

In an embodiment, at least one of these bioreactors comprises a concentrator module as hereinabove described.

Claims

CLAIMS1. A concentrator module to receive a sample comprising biological material in a fluid buffer medium and increase the concentration of a target biological material present within the sample comprises a microfluidic apparatus including: an inlet, an output outlet, at least one further outlet, and a fluid pathway therebetween including a processing unit that is operable to preferentially divert the target biological material to the output outlet, and for example preferentially diverts buffer medium/ undesired solid material/ non-target biological material to the further outlet, for example to be discarded or further processed; wherein the processing unit includes elements disposed to operate on both of the principles of dielectrophoresis (DEP) and alternating current electroosmosis (ACEO).
2. The concentrator module of claim 1, wherein the processing unit comprises at least a microfluidic ACEO device and a microfluidic DEP device fluidly downstream of the ACEO device.
3. The concentrator module of claim 1 or claim 2, wherein the target biological material comprises bacterial material and/ or viral material.
4. The concentrator module of any preceding claim, wherein the processing unit of the concentrator module comprises a first series of microfluidic pathways each configured for the passage of fluid through one or more microfluidic ACED devices and a second series of microfluidic pathways each configured for the passage of fluid through one or more microfluidic DEP devices
5. The concentrator module of any preceding claim, wherein the microfluidic ACEO device comprises an ACE) electrode system disposed in parallel to a flow direction through the device, and for example comprises an ACED electrode system disposed on one or more sides of a flow channel in the device.
6. The concentrator module of claim 5, wherein the processing unit of the concentrator module comprises an upstream microfluidic ACED device having an electrode system configured to be operable to tend to draw biological material flowing through a channel in the device towards one or more sides of the channel and a downstream microfluidic DEP device having an electrode system juxtaposed therewith and operable to tend to act on larger particles thereby drawn into its vicinity.
7. The concentrator module of claim 6, comprising a microfluidic ACEO device having a parallel ACED electrode system and a microfluidic DEP device having a DEP electrode system are positioned fluidly consecutively on the same side of a microfluidic channel.
8. The concentrator module of one of claims 5 to 7, wherein a flow channel within the microfluidic concentrator module divides downstream of the microfluidic ACED device to define two onward flow channels, a first onward flow channel being configured such that it receives a flow from that part of the undivided channel in the vicinity of the ACED electrode system and a second onward flow channel being configured such that it receives a flow from away from the vicinity of the ACED electrode system.
9. The concentrator module of claim 8 comprising: an inlet flow channel in fluid communication with a microfluidic ACED device comprising one or more microfluidic channels defining an initial flow direction through the device and an ACED electrode system disposed in parallel to the flow direction, and operable to tend to cause biological material to segregate into a first part of the flow channel; a divide in the inlet flow channel into a first onward flow channel dividing from the same in such manner as to be fluidly continuous with the first part of the flow channel, and a second onward flow channel; the second onward flow channel optionally continuing to a discharge outlet; the first onward flow channel continuing successively to and in fluid communication with the microfluidic DEP device.
10. The concentrator module of claim 8 or 9, wherein the two onward flow channels comprise parallel flow channels and for example respective upper and lower parallel flow channels.
11. The concentrator module of any preceding claim, operable to separate a final output into which larger particles comprising a first target material are separated and concentrated and sent to a first target outlet, and a first output comprising the rest of the sample.
12. The concentrator module of claim 11, comprising a further microfluidic processing unit that is operable to process the first output and to preferentially divert a further target biological material to a second output outlet.
13. The concentrator module of any preceding claim, comprising a secondary microfluidic DEP device provided upstream of the microfluidic ACEO device and the microfluidic DEP device and configured to be operable to generate a negative DEP such as to tend to urge material towards a region of vortex generation of the principal microfluidic ACED device.
14. A concentrator module to receive a sample comprising biological material in a fluid buffer medium and increase the concentration of a target biological material present within the sample comprises a microfluidic apparatus including: an inlet, an output outlet, at least one further outlet, and a fluid pathway therebetween including a processing unit that is operable to preferentially divert the target biological material to the output outlet, and for example preferentially diverts buffer medium/ undesired solid material/ non-target biological material to the further outlet, for example to be discarded or further processed; wherein the processing unit includes a microfluidic ACED device comprising one or more microfluidic channels defining an initial flow direction through the device and an ACED electrode system disposed at an angle to the flow direction.
15. The concentrator module of claim 14, wherein the microfluidic ACEO device comprises one or more inlet microfluidic channels defining an initial flow direction through the device and an ACEO electrode system disposed at an angle to the flow direction electrode system comprising a plurality of parallel electrodes at an angle to the initial flow direction.
16. The concentrator module of claim 14 or 15, wherein the electrode angle is 2to 35 degrees to a direction parallel to the initial flow direction through the device.
17. The concentrator module of one of claims 14 to 16 comprising a further microfluidic DEP device provided upstream of the microfluidic ACEO device and configured to be operable to generate a negative DEP such as to tend to urge material towards a region of vortex generation of the microfluidic ACEO device.
18. A concentrator module to receive a sample comprising biological material in a fluid buffer medium and separate and increase the concentration of a first and a second target biological material present within the sample comprising a microfluidic apparatus including fluidly in series the elements of a module according to one of claims 1 to 13 and of a module according to one of claims 14 to 17.
19. A concentrator module to receive a sample comprising biological material in a fluid buffer medium and separate and increase the concentration of a first and a second target biological material present within the sample comprising a microfluidic apparatus including fluidly in series: an inlet; an inlet flow channel in fluid communication with a first microfluidic ACEO device comprising one or more microfluidic channels defining an initial flow direction through the device and an ACED electrode system disposed in parallel to the flow direction, and operable to tend to cause biological material to segregate into a first part of the flow channel; at least first part of the flow channel being fluidly continuous with an onward flow channel continuing successively to and in fluid communication with a microfluidic DEP device and a second microfluidic ACEO device comprising one or more microfluidic channels defining an initial flow direction through the device and an ACEO electrode system disposed at an angle to the flow direction; selectively closable first and second separation channels into which the onward flow channel divides fluidly downstream of the microfluidic DEP device; the first separation channel continuing to a first target material outlet; the second separation channel continuing to a second target material outlet.
20. A concentrator module according to claim 19, wherein the inlet flow channel is divided downstream of the first microfluidic ACEO device into a first onward flow channel dividing from the same in such manner as to be fluidly continuous with the first part of the flow channel, and a second onward flow channel; the second onward flow channel optionally continuing to a discharge outlet; the first onward flow channel continuing successively to and in fluid communication with the microfluidic DEP device and the second microfluidic ACEO device.
21. A microfluidic system comprising a plurality of fluidly connected microfluidic chambers, each microfluidic chamber comprising: a fluid sample inlet; a fluid sample outlet; a selectably closable valve operable to enable gas to be vented from the chamber; a pressurisation system operable to apply an overpressure to one or more first microfluidic chambers being fluidly most upstream, wherein at least one of the microfluidic chambers comprises a concentrator module in accordance with any one of claims 1 to 20.
22. A method to process a sample comprising biological material in a fluid buffer medium and increase the concentration of at least one target biological material present within the sample comprises passing the biological material and fluid buffer medium through a concentrator module in accordance with any one of claims 1 to 20 to increase the concentration of the biological material in an output stream.