WO2016116790A1

WO2016116790A1 - Microdialysis device

Info

Publication number: WO2016116790A1
Application number: PCT/IB2015/059719
Authority: WO
Inventors: Frederick Kiguli BALAGADDÉ; Brilliant Bonisile LUTHULI
Original assignee: Kwazulu-Natal Research Institute For Tb-Hiv (k-Rith) Npc
Current assignee: Kwazulu-Natal Research Institute For Tb-Hiv (k-Rith) Npc
Priority date: 2015-01-22
Filing date: 2015-12-17
Publication date: 2016-07-28
Anticipated expiration: 2017-07-22
Also published as: ZA201704564B

Abstract

This invention relates to microfluidics and provides a microfluidic device comprising a fluid flow circuit that includes an array of microfluidic chemical reaction chambers (100) with differing internal volumes, the reaction chambers (100) being configured to receive a chemical- containing fluid by way of the fluid flow circuit and to retain the fluid for the duration of all or part of a chemical reaction involving one or more of the chemicals contained in the fluid. The reaction chambers (100) are configured in groups in which each reaction chamber in a group has a similar volumetric size to the other reaction chambers in that group of reaction chambers (100).

Description

lCRODIALYSIS DEVICE

Field of the invention

[001 ] This invention relates to analytical chemistry using microfluidics, chemical assays and more specifically bioassays.

[002] In particular, the invention relates to microfluidic devices and processes for adding or removing substrates (for instance reagents, such as drugs and growth factors) to or from a captive microbial population using such microfluidic devices and processes.

Background to the invention

[003] Microfluidics is a multidisciplinary field involving engineering, physics, chemistry, biochemistry, nanotechnology and biotechnology. Microfluidics deals with the manipulation of liquids within containers and channels of submillimeter size— for purposes of this specification, the technical differences between nanofluidics, microfluidics and millifluidics are not relevant and all of these disciplines will be treated as microfluidics. Microfluidics can be considered both as a science (the study of the behaviour of fluids in micro-channels) and a technology (the manufacture of microfluidics devices for applications such as lab-on-a-chip devices) .

[004] Microfluidic devices find increasing application in analytical chemistry and in systems for applied analytical purposes in chemistry, biochemistry, and life science in general. All-in-one microfluidic chips capable of separation, reaction, and detection have been developed that constitute microscale total analysis systems ( TAS) or lab-on-a-chip devices. These integrated chips utilise scalable fabrication techniques and have the potential to automate chemical analysis, particularly point- of-care, clinical, and medical diagnostics.

[005] The devices are designed to handle small fluid volumes and to operate within the microliter to picoliter fluid volume range (1 microliter = 1 l O⁶ liter; 1 picoli†er=l ΐ θ¹² liter). Natural microchannels, such as blood vessels and plant capillary vessels, are mostly excluded from microfluidics, except to the extent that microfluidic devices are configured to mimic such natural microchannels in shape and/or function. Microfluidic devices can be engineered to provide control over many system parameters. In polydimethylsiloxane-based devices (PDMS devices) for instance, membrane microvalves can be incorporated to provide the required control, thereby providing the means to realise microfluidic/large scale integration. A basic PDMS microfluidic device is composed of two elastomer layers. One layer (the flow layer) typically contains channels for flowing liquids and the other layer (the control layer) contains channels that deflect the membrane valve into the flow channel and stop liquid flow when pressurised with air or liquid. Where a control channel and a flow channel cross, a valve is created. The thin membrane separating the two channels deflects into the flow channel when the control channel is pressurised, creating a complete seal.

[006] The hydraulic devices incorporated in a hydraulic microcircuit enable the implementation of predetermined functions on the device. Typically fluids are moved, mixed, separated or otherwise processed. Numerous applications employ passive fluid control techniques like capillary forces. In some applications however, external actuation means are used for a directed transport of the fluids on the device, including circuit switching, mixing, pumping, redirecting and facilitating chemical reactions within microscale reaction chambers formed in the device. This network of microchannels is connected to the outside by various macro- world interfaces, including inputs and outputs pierced through the device that allow the injection or removal of liquids or gas, typically through tubing, syringe adapters or even free holes in the device.

[007] The bioassay processes of the invention are preferably implemented on microfluidic devices colloquially known as lab-on-a-chip devices. The word "chip" in lab-on-a-chip stems from the original fabrication method, a modified form of photolithographic etching used to manufacture computer microchips, which allows control of surface feature shapes and sizes on the same scale (nm to m) that living cells sense and respond to in their natural tissue milieu. In the exemplary embodiments of the invention described below, the microfluidic systems are made by etching microfluidic channel and chamber patterns photolithographically onto a silicon substrate. The etched pattern serves as a negative for a moulding made on the substrate from a biocompatible material such as polydimethylsiloxane (PDMS) that is allowed to polymerise on the substrate, essentially creating a positive of the negative mould onto the silicon.

[008] The invention is not restricted to PDMS-based devices and the microfluidic devices of the invention can be fabricated out of other materials, such as silicon, plastic, glass, silk or the like using micromoulding, microetching, laser etching, injection moulding, photo polymerisation, solid object printing and other microscale manufacturing approaches and the invention is not restricted to PDMS-based devices.

[009] The term "bioassay" is often used as shorthand for biological assays or assessments. These are scientific experiments typically involving the use of live or extracorporeal animal or plant material to determine a biological activity, such as the biological activity of a substance such as a hormone or drug. [0010] In medicine, dialysis typically refers to the processes involved in the removal of waste and water from the blood and is used primarily as an artificial replacement for lost kidney function in people with renal failure. However, in chemistry dialysis refers more generally to the separation of substances in solution by means of unequal diffusion, normally through semipermeable membranes. Unless the context clearly indicates otherwise, it is in this more general sense that the terms "dialysis" and "dialyser" are used in this specification. It must also be pointed out that the microdialyser of the invention is intended to function largely as an extracorporeal microbioassay device and is unrelated to the microdialysis probes normally used in minimally invasive medical sampling techniques.

Summary of the invention

[001 1 ] According to this invention a microfluidic device comprises a fluid flow circuit that includes an array of microfluidic chemical reaction chambers with differing internal volumes, the reaction chambers being configured to receive a chemical-containing fluid by way of the fluid flow circuit and to retain the fluid for the duration of all or part of a chemical reaction involving one or more of the chemicals contained in the fluid.

[0012] Each reaction chamber may be vol u metrically different from each other reaction chamber, but in the preferred form of the invention, the reaction chambers are conveniently configured in groups in which each reaction chamber in a group has a similar volumetric size to the other reaction chambers in that group of reaction chambers.

[0013] The microfluidic reaction chambers are preferably individually controllable, the fluid flow circuit including flow channels in valve- controlled fluid flow communication with the reaction chambers, the valves being individually controllable. The valves are preferably controlled by the inclusion, in the microfluidic device, of a control fluid circuit including a plurality of individually controllable valves configured to open and close one or more of the flow channels and reaction chambers.

[0014] Assays, typically, are procedures by which the properties of substances are measured. In the implementations of the invention contemplated in this specification however, the term "assay" is preferably (but not necessarily) intended to refer to an investigative, analytic procedure in life sciences, including for instance, medicine, pharmacology, biology and environmental science. Such assay procedures typically involve assessing or measuring the reaction of a target entity (the analyte) in the presence of an exogenous reactant (the reagent), the outcome of the assay being the functional activity or reaction of the analyte to the reagent.

[0015] The microfluidic device of the invention is preferably implemented as a microfluidic chemical assay system in which the microfluidic reaction chambers are configured to permit simultaneous, parallel chemical analyses of an externally supplied analyte constituted by the chemical- containing fluid, the fluid flow circuit being configured to permit the introduction of the analyte to one or more predetermined reaction chambers.

[001 6] In this embodiment of the invention, the microfluidic reaction chambers of the microfluidic device are preferably configured to form part of an analyte fluid flow circuit by means of which the analyte may be introduced into all or some of the microfluidic reaction chambers, the microfluidic device including a separate reagent fluid flow circuit that is configured to permit the introduction of an externally supplied reagent into all or some of the microfluidic reaction chambers.

[001 7] In a preferred form of this embodiment of the invention, the analyte circuit conveniently includes flow channels that are in fluid flow communication with valve-controlled individually controllable reaction chambers and the reagent flow circuit includes flow channels in fluid flow communication with a plurality of valve-controlled individually controllable microfluidic reagent chambers that are in valve-controlled fluid flow communication with the reaction chambers, the reagent circuit being configured to supply the reagent first to the reagent chambers and then to the reaction chambers.

[0018] In this embodiment of the invention, valve control may conveniently be provided by the inclusion of a control fluid circuit including a plurality of individually controllable valves configured to open and close any one or more flow channels, reaction chambers and reagent chambers.

[0019] To enable the inclusion of fluid flow and control fluid circuits in a single device, the microfluidic device of the invention is conveniently implemented in polydimethylsiloxane (PDMS), the microfluidic device comprising a plurality of PDMS elastomer layers including one or more flow layers including the flow channels, microfluidic reaction chambers and microfluidic reagent chambers forming part of the analyte and reagent circuits, and one or more control layers including a plurality of individually controllable membrane valves interconnected by means of individually controllable control channels, the membrane valves being configured to be deflected selectively into a flow channel to enable or prevent fluid flow within the corresponding flow channel when the control channel is pressurised with a pressure fluid.

[0020] The valves, circuits and chambers are "addressed" and controlled by means of control valves integrated in the microfluidic circuitry of the microdialyser and connected to macro-scale fluid inputs and outlets by means of macro-scale connectors, the macro-scale devices being driven either manually or by way of computer programs.

[0021 ] The fluid flow channels may include a combinatorial mixer or mixers and inlet and outlet ports suitable to allow interconnection of the microfluidic device into such macro-scale fluid flow and control circuits.

[0022] Macrophages are a type of white blood cell that engulfs and digests cellular debris, foreign substances, microbes, and cancer cells in a process called phagocytosis. Some pathogens subvert this process and instead live inside the macrophage. Diseases with this type of behaviour include tuberculosis which is caused by Mycobacterium tuberculosis— once engulfed by a macrophage, the causative agent of M. tuberculosis avoids cellular defenses and uses the cell to replicate.

[0023] Current tuberculosis drug susceptibility tests rely on the assumption that culturing patient-derived mycobacteria in drug-containing medium in vitro is a reliable predictor of in vivo drug efficacy. This assumption overlooks the volume difference between the in vitro (test tubes and petri dishes) and in vivo (macrophages and granulomatous lesions) tuberculosis growth environments and their respective implications for drug tolerance. However, unlike the mycobacteria in freshly inoculated conventional (macro-scale) cultures, a single mycobacterium growing within a macrophage experiences a confined growth environment that the applicant believes might induce drug tolerance and to the degree that confinement induces drug tolerance, current drug susceptibility tests are poor predictors of in vivo drug efficacy.

[0024] In a specific application of this form of the invention, the microfluidic device can be configured as a microfluidic bioassay system directed at investigating space-confinement-induced drug tolerance in mycobacteria as a possible underlying mechanism for macrophage- induced drug tolerance and persistence, for instance, the phenomenon whereby Mycobacterium tuberculosis cells growing in human tissues survive prolonged exposure to drugs even though the same drugs are capable of rapidly sterilising genetically identical mycobacteria growing in axenic cultures.

[0025] In a preferred assay system embodiment of the invention, the microfluidic device conveniently configured as a microfluidic bioassay system implemented as a cell culture model configured to analyse cellular behaviour in a regime where volume confinement induces behaviours in the cells that are not normally encountered in macro-scale culture settings. The cells may be prokaryotes or eukaryotes.

[0026] In this embodiment of the invention, the reaction chambers of the microfluidic device are preferably configured in a plurality of sizes based on the approximately 5 picoliter (0.005nL) volume of the membrane- bound compartment of the average human alveolar macrophage.

[0027] The reaction chambers may be configured in a range of sizes, from a practically attainable size (given current fabrication limitations) approaching 0.005nL to 2nL.

[0028] More specifically, the reaction chambers may be configured in a range of sizes from 0.22nL to 1 .7nL and preferably in the following range of sizes: 0.22nL; 0.5nL; 1 .2nL; 1 .7nL.

[0029] Besides the physical structure of the reaction chambers, the fluid exchange characteristics of the microfluidic device are preferably configured, in a preferred form of this embodiment of the invention, to approximate in vivo diffusive medium exchange.

[0030] In such an embodiment, the fluid flow channels and particularly the fluid flow channels between the microfluidic reagent and reaction chambers are preferably dimensioned†o restrict fluid exchange between the reagent and reaction chambers to passive diffusive exchange. For instance and using the example of a mycobacterial cell culture inoculated in the reaction chamber and a growth medium introduced into the reagent chamber, the fluid flow channels between the chambers are preferably dimensioned to ensure that nutrient molecules diffuse from the reagent chamber into the reaction chamber and mycobacterial metabolic waste products diffuse from the reaction chamber into the reagent chamber down their respective concentration gradients.

[0031 ] In this embodiment, the fluid flow channels are preferably controlled such that the reagent and reaction chambers are at atmospheric pressure whenever the valves between the chambers are opened.

[0032] The invention includes a method of modelling persistence in a microbial analyte in a volume-constrained growth environment using the microfluidic device of the invention, the method including the steps of: inoculating one or more reaction chambers of the microfluidic device with the microbial analyte; charging the inoculated reaction chamber/s with a growth medium within which to culture the microbial analyte mixed with a reagent constituted by an antimicrobial agent; and culturing the microbial analyte in the growth medium and recording growth or the absence of growth of the microbial analyte in the reaction chamber/s and, in particular, variations in growth rate, if any, as a function of reaction chamber volume.

[0033] The invention includes an additional method of analysing persistence in a microbial analyte in a volume-constrained growth environment using the microfluidic device of the invention, the method including the steps of: inoculating one or more reaction chambers of the microfluidic device with the microbial analyte; charging the inoculated reaction chamber/s with growth medium free of antimicrobial agents within which to culture the microbial analyte; culturing the microbial analyte in the growth medium and recording growth or the absence of growth of the microbial analyte and, in particular, variations in growth rate, if any, as a function of reaction chamber volume; and replacing the drug-free growth medium in the inoculated reaction chamber/s with a similar growth medium including an antimicrobial agent and recording growth or the absence of growth of the microbial analyte and, in particular, variations in growth rate, if any, as a function of reaction chamber volume.

[0034] As a last step in the method outlined immediately above, replacing the drug-containing growth medium in the inoculated reaction chamber/s with drug-free growth medium and recording growth or the absence of growth of the microbial analyte and, in particular, variations in growth rate, if any, as a function of reaction chamber volume.

[0035] The invention further includes a method of resolving the bacteriostatic and bactericidal credentials of an antimicrobial agent using the microfluidic device of the invention, the method including the steps of: inoculating one or more reaction chambers of the microfluidic device with a microbial analyte; charging the inoculated reaction chamber/s with a growth medium within which to culture the microbial analyte mixed with a reagent constituted by an antimicrobial agent; culturing the microbial analyte in the growth medium and observing the progress of microbial growth, if any, in the reaction chamber/s; recording growth or the absence of growth of the microbial analyte in the reaction chamber/s and designating the microbial analyte in any chamber/s showing growth of the microbial analyte as being resistant to the antimicrobial agent; replacing the inoculated reaction chamber/s with a similar growth medium excluding the antimicrobial agent; recording the resurgence, if any, of growth of the microbial analyte in the reaction chamber/s; and in a final record, recording the antimicrobial agent as bacteriostatic in respect of the microbial analyte in any chamber/s showing resurgent growth and bactericidal in respect of the microbial analyte in any chamber/s in which growth recovery is observed.

[0036] Resurgence of microbial growth under reagent-free culture implies that the antimicrobial agent applied during the first microdialysis step is bacteriostatic since it failed to eradicate or kill the microbes. Conversely, the absence of growth recovery implies that the antimicrobial agent was bactericidal, given that it rendered the microbes nonviable. Brief Description of the drawings

[0037] The invention will be further described with reference to the accompanying drawings in which:

Figures 1 and 2 are diagrammatic representations of a reaction chamber assembly forming part of the microdialyser of the invention;

Figures 3 and 4 are diagrammatic representations of an alternative reaction chamber assembly for the of the invention;

Figure 5 is an optical micrograph of part of one embodiment of a lab-on-a-chip microdialyser according to the invention;

Figure 6 is an enlargement of a portion of Figure 5; and

Figure 7 is a diagrammatic representation of a second embodiment of a lab-on-a-chip microdialyser according to the invention.

Description of embodiments of the invention

[0038] The invention will be illustrated and described with reference to a microfluidic device implemented as a microdialyser. The nature and operation of the microdialyser will be demonstrated and described with reference to two specific applications: an application directed at resolving the bacteriostatic and bactericidal credentials of antimicrobial agents; and an application directed at investigating space-confinement- induced drug tolerance a possible underlying mechanism for macrophage-induced drug tolerance and mycobacterial growth in the macrophage.

[0039] It will be appreciated that these applications of the microdialyser are highlighted purely as a means of illustrating and exemplifying the invention and is not intended to restrict the invention to such a microdialyser.

[0040] Microfluidic devices can be engineered to provide control over many system parameters. Typically, the devices are fabricated to constitute entire hydraulic microcircuits, complete with hydraulic control devices configured to implement one or more predetermined functions on the device. Fluids can be moved, mixed, separated or otherwise processed.

[0041 ] Most applications employ a combination of passive and driven fluid flow and control techniques. The former techniques typically employ capillary forces, whilst the latter techniques employ external actuation means for directed transport of the fluids on and through the device, including pumping, circuit switching, mixing, redirecting and facilitating chemical reactions within microscale reaction chambers formed in the device, typically with the aid of externally derived pressure fluids such as pressurised liquids or compressed gases. To facilitate such external actuation and to provide microscale/large-scale integration, the network of microchannels on the device is normally connected to the outside by various macro-world interfaces, including inputs and outputs pierced through the device that allow the injection or removal of liquids or gas, typically through tubing, syringe adapters or free holes in the device.

[0042] In PDMS-based devices, the functionality described above is implemented and obtained by means of a number of elastomer layers including one or more flow layers in which flow channels are formed for flowing liquids and one or more control layers in which control channels are formed. The microdialyser of the invention is preferably implemented in a PDMS device such as this.

[0043] In the microdialyser of the invention at least two flow circuits are provided, the first flow circuit constituting an analyte circuit that includes flow channels that are in fluid flow communication with a plurality of individually controllable microscale reaction chambers, the analyte circuit being configured to inoculate the reaction chambers with cell cultures or some other analyte. The second flow circuit is a reagent circuit that includes flow channels in fluid flow communication with a plurality of individually controllable microscale reagent chambers that are in valve- controlled fluid flow communication with the reaction chambers. The reagent circuit is configured to supply a reagent or reagents first to the reagent chambers and, depending on valve control, also to the reaction chambers. The reagent will be selected for suitable reaction with the analyte and may include a drug or an antimicrobial adjuvant with or without other reagents, such as cell culture growth medium.

[0044] In addition, the device includes one or more control layers formed with individually controllable membrane valves interconnected by means of individually controllable channels that deflect the membrane valves selectively into a flow channel to enable or prevent liquid flow within the flow channel when the control channel is pressurised with a pressure fluid.

[0045] Fluid flow control in the microdialyser of the invention is preferably achieved by means of individually addressable and controllable valves and valve-controlled fluid flow circuits and microfluidic chambers. The valves, circuits and chambers, typically, will be "addressed" by means of control valves integrated in the microfluidic circuitry of the microdialyser and connected to macro-scale fluid inputs and outlets by means of macro-scale connectors. The macro-scale inputs and outlets, typically, will be controlled by a computer program. To this end, the flow channels may include a combinatorial mixer or mixers and inlet and outlet ports suitable to allow interconnection of the microdialyser into macro-scale liquid and control fluid flow circuits. This facilitates integration of the microfluidic circuits of the microdialyser into a macro-scale chemical assay or bioassay that is programmable to process biochemical and chemical reactions and to integrate these reactions in predetermined workflows, assays and analyses.

[0046] Figures 1 to 4 illustrate a single microscale reaction or growth chamber— Figures 1 and 2 illustrate a first embodiment of the microscale reaction chamber 100 and Figures 3 and 4 illustrate a second embodiment of the microscale reaction chamber 100.1 .

[0047] In the applications selected to illustrate the invention, the microscale reaction chambers 100, 100.1 operate as cell culture chambers. In the preferred form of the invention the microdialyser includes an array of reaction chambers 100, 100.1 that range in size from 0.22nL through 0.5nL and 1 .2nL to 1 .7nL. The microscale reaction chambers 100, 100.1 are each provided with a conditioning chamber 102, 102.1 that is in fluid communication with the reaction chamber 100, 100.1 across an inlet/outlet diffusion channel 104, 104.1 .

[0048] In the embodiment illustrated in Figures 3 and 4, the conditioning chamber 102.1 is concentric to and extends about the reaction chamber 100.1 and it is connected to the conditioning chamber 102.1 across multiple diffusion channels 104.1 . The diffusion channels 104 can be opened or closed by means of separately addressable membrane-type reaction chamber valves 106, 106.1 . The conditioning chamber 102, 102.1 is connected to a medium flow input channel 108, 108.1 that is supplied with a cell growth medium from an external supply. The conditioning chamber 102, 102.1 is also connected to a medium flow output channel 1 18, 1 18.1 that leads to an external waste outflow. From the medium flow input channel 108, 108.1 growth medium is pumped into the conditioning chamber 102, 102.1 through separately addressable inlet supply channels 1 10, 1 10.1 , each controlled by means of a membrane-type valve 1 12, 1 12.1 and out through separately addressable outlet channels 1 14, 1 14.1 , each controlled by means of a membrane-type valve 1 16, 1 16.1 . Similarly, the reaction chamber 100, 100.1 is connected to a flow input channel 124, 124.1 that is supplied with a mycobacterial cell suspension from an external supply. The reaction chamber 100, 100.1 is also connected to a cell suspension output channel 130, 130.1 that leads to an external waste outflow.

[0049] From the cell suspension input channel 124, 124.1 , the cell suspension is pumped into the reaction chamber 100, 100.1 through a separately addressable inlet supply channel 122, 122.1 controlled by means of a membrane-type valve 120, 120.1 . The cell suspension can also be pumped out of the reaction chamber 100, 100.1 through a separately addressable outlet channel 128, 128.1 , controlled by means of a membrane-type valve 126, 126.1 .

[0050] When the reaction chamber valves 106, 106.1 are opened, passive diffusive exchange occurs between the growth chambers 100, 100.1 and the conditioning chambers 102, 102.1 . Nutrient molecules diffuse from the conditioning chamber 102, 102.1 into the reaction chamber 100, 100.1 while mycobacterial metabolic waste products diffuse from the reaction chamber 100, 100.1 into the conditioning chamber 102, 102.1 .

[0051 ] The microdialyser uses a microdialysis scheme periodically to add fresh nutrients and remove waste from the captive population of mycobacteria in the reaction chambers 100, 100.1 through diffusive exchange, mimicking the passive bidirectional exchange of pro- and anti- mycobacterial factors across a macrophage membrane. [0052] Periodically (for instance, hourly), the reaction chamber valves 106, 106.1 are opened in microdialysis cycles lasting approximately 60 seconds each to allow the relatively small molecules of the growth medium and mycobacterial metabolic waste products to diffuse freely between the two chambers down their respective concentration gradients— fresh nutrients diffuse into the reaction chamber 100, 100.1 while mycobacterial metabolic waste products diffuse into the conditioning chamber 102, 102.1 . The relatively large size and non-motility of the mycobacterial cells deters the cells from exiting the reaction chamber 100, 100.1 when the reaction chamber valves 106, 106.1 are opened.

[0053] Next, the reaction chamber valves 106, 106.1 are closed and the conditioning chamber valves 1 12, 1 12.1 and 1 16, 1 16.1 are opened to flush the conditioning chamber 102, 102.1 and to refill the chamber 102, 102.1 with fresh medium to await the next microdialysis step.

[0054] In-between consecutive microdialysis steps, the conditioning chamber 102, 102.1 can be filled with phosphate buffer saline (PBS) solution to discourage microbes from growing inside these chambers.

[0055] Using colorimetric assays, the applicant found that on average, the microdialyser converted the reaction chamber fluid within six microdialysis steps. The number of steps may be increased (or reduced) by opening the diffusion channels 104, 104.1 for a shorter (or longer) duration during microdialysis.

[0056] The diffusion channels 104, 104.1 are dimensioned to permit relatively free flow of the growth medium into and out of the reaction chamber 100, 100.1 and no attempt is made to select the size of the diffusion channel to mimic membrane transport or to dimension the diffusion channel 104, 104.1 such that it constitutes or mimics a selectively permeable membrane for purposes of diffusion. However, during the microdialysis process, microbes are prevented from crossing the channels 104, 104.1 from the reaction chambers 100, 100.1 into the conditioning chambers 102, 102.1 by precluding fluid flow currents between the reaction chambers 100, 100.1 and the conditioning chambers 102, 102.1 when the valves 106, 106.1 are open. This is accomplished by maintaining a zero differential pressure between the reaction chambers 100, 100.1 and the conditioning chambers 102, 102.1 .

[0057] In this process, when the microbial cells are introduced into the reaction chambers 100, 100.1 at the beginning of the assay, the inlet channels 122, 122.1 connecting the reaction chamber 100, 100.1 to the pressurized bacterial suspension input flow line 124, 124.1 are closed first while the outlet channels 128, 128.1 connecting to the outlet 130, 130.1 (which is maintained at atmospheric pressure) are still open. Thus when the outlet channels 128, 128.1 are closed a few seconds later, the pressure in the reaction chamber is equal to atmospheric pressure, where it is maintained throughout the subsequent measurements. Similarly, during each microdialysis step, once the conditioning chambers 102, 102.1 is filled with fresh fluid, the channels 1 10, 1 10.1 connecting the conditioning chamber 102, 102.1 to the pressurized medium inlet 108, 108.1 are closed first while the channels 1 14 connecting to the outlet 1 18, 1 18.1 (which is maintained at atmospheric pressure) are still open. Thus when the outlet channels 1 14, 1 14.1 are closed a few seconds later, the pressure in the conditioning chamber 102, 102.1 is equal to atmospheric pressure.

[0058] Microbial displacements due to gravitational forces are negligible given the shallow, planar structure of the microdialyser system. In the absence of gravitational or fluid-flow driven forces, perturbation of the microbial cells due to external forces is precluded.

[0059] Because the cells themselves are non-motile, they remain in the reaction chamber 100, 100.1 during microdialysis. Nevertheless, partial cell loss due to cellular motility is possible, but it at a scale that is unlikely to affect the outcome of any experiment.

[0060] Figure 5 is an optical micrograph of the lab-on-a-chip microdialyser system 200 showing a single 30-reac†or module (outlined— 250). The reactor module 250 consists of a plurality of individually addressable micro-reaction chambers 100.3 similar to the micro-reaction chambers 100.3 described with reference to Figures 1 to 4. The micro- reaction chambers 100.3 are arranged in an array as follows: 6 x 0.22nL reaction chambers; 8 x 0.5nL reaction chambers; 8 x 1 .2nL reaction chambers; 6 x 1 .7nL reaction chambers

[0061 ] The device features 5 cell input ports 252, 8 medium input ports 254 and 10 output ports 256, the latter connected to one another by way of medium flow channels similar to the medium flow channels 108, 108.1 ; 1 18, 1 18.1 ; 124, 124.1 ; and 130, 130.1 described with reference to Figures 1 to 4.

[0062] The reaction chambers 100.3 are inoculated with mycobacterial cells introduced to the device by way of cell suspension input ports 252.

[0063] Figure 6 illustrates (in Figures 6A, 6B and 6C) three stages of the microdialysis scheme of the device of Figure 5. Each of Figures 6A, 6B and 6C is an enlargement of the same section of the Figure 5 optical micrograph illustrating, in various stages of operation (6A, 6B, 6C), a portion of the reactor module 250 (the portion outlined and numbered 6 in Figure 5). The reactor module section 6 shows 3 reaction chambers 100.3, each having an associated conditioning chamber 102.3 extending about the reaction chamber 100.3.

[0064] In Figure 6A the middle reaction chamber 100.3 is isolated from the conditioning chamber 102.3 extending about the reaction chamber 100.3. In this state, the diffusion channel 104.3 is closed by means of the reaction chamber valve . In Figure 6B, in a first microdialysis step, the reaction chamber valve is opened, thereby opening the diffusion channel 104.3 to allow diffusive fluid exchange between the reaction chamber 100.3 and the conditioning chamber 102.3. After a suitable delay (typically 60 seconds), the reaction chamber valve is closed and the conditioning chamber 102.3 is flushed and refilled with growth medium, whereupon the microdialysis step is repeated. After a series of microdialysis steps, the fluid in the reaction chamber 100.3 is completely replaced with the fluid introduced via the conditioning chamber, as illustrated in Figure 6C.

[0065] In addition to mycobacterial inoculation and growth medium supply, the microdialyser 200 also includes a charging, mixing and flow system for drug formulations. The drug formulation flow system includes a combinatorial mixer or formulator 258. This allows interconnection of the microdialyser 200 into macro-scale liquid flow and control fluid flow circuits, thereby facilitating integration of the microfluidic circuits of the microdialyser 200 into a macro-scale chemical assay or bioassay that is programmable to process biochemical and chemical reactions and to integrate these reactions in predetermined workflows, assays and analyses.

[0066] Since PDMS is optically transparent, cell growth in the reaction chambers 100.3 can be monitored individually by optical microscopy to provide automated, real-time, non-invasive measurement of cell density.

[0067] The system is operated in two alternating states: microdialysis and cell culture incubation.

[0068] During microdialysis, the conditioning chamber 102.3 is filled with fresh growth medium (Figure 6A). Next, the reaction chamber valves connecting the conditioning (102.3) and reaction chambers (100.3) are opened for 60 seconds to allow small molecules to freely diffuse between the two chambers down their respective concentration gradients, thereby allowing fresh nutrients to diffuse into the reaction chamber 100.3 while bacterial metabolic waste products diffuse into the conditioning chamber 102.3 (Figure 6B).

[0069] Although the comparatively large size of bacteria deters the microbes from exiting the reaction chamber during microdialysis, a few cells may occasionally spill into the conditioning chamber. Nevertheless, observations suggest that this effect is minor and does not interfere with the microdialysis or culture scheme. This notwithstanding, a more appropriate protocol is to rinse the conditioning chamber 102.3 with a lysis buffer, a wash buffer, growth medium or a combination of the aforementioned after microdialysis to flush any bacteria that may spill into the conditioning chamber during microdialysis and to discourage bacterial growth in the conditioning chamber.

[0070] During cell culture incubation, bacteria within the reaction chamber 100.3 are incubated for approximately 1 hour (or a more appropriate period of time) in fluidic isolation from the conditioning chamber 102.3 (that is with the reaction chamber valve closed— Figure 6C).

[0071 ] The microdialyser can facilitate non-invasive addition or removal of drugs and other growth factors from mycobacterial populations confined within the reaction chambers 100.3.

[0072] The microdialyser can be used in a number of applications, but as indicated above, one exemplary application is in resolving the bacteriostatic and bactericidal credentials of antimicrobial agents. [0073] Antibiotic drug therapy and immune system control of bacterial infections can take the form of bacteriostatic or bactericidal action. In the case of many bacterial infections, bactericidal action is a key element to a sterilizing strategy. Routine drug susceptibility testing can be cumbersome for distinguishing between bacteriostatic and bactericidal action of drugs. The most frequently used method for distinguishing between bactericidal and bacteriostatic antimicrobial compounds is the so-called "cell washing method". In this method, bacterial cells are grown to an appropriate cell density. A test drug is added to the cell culture at a specified concentration and the effect on the microbial population is analysed. The microbes are subsequently washed by centrifugation to remove the drug and then re-suspended in drug-free medium. The culture is then analysed for signs of continuing microbial growth if any. This method is laborious and impractical for routine use in clinical drug susceptibility testing. It also consumes a lot of time and large volumes of reagents and is thus expensive, besides being invasive, since the centrifugation/wash steps could harmfully affect the growth of the microbial cells.

[0074] The microdialyser of the invention resolves the bacteriostatic and bactericidal credentials of antimicrobial agents rapidly using a two-step microdialysis process.

[0075] In a first step of this process, the reaction chambers 100.3 of the microdialyser that are inoculated with microbes and charged with growth medium within which the microbes are cultured. The antibacterial agent being tested is mixed in with the growth medium. If the microbes continue to multiply in the drug-containing medium, the microbes are scored as resistant to the antibacterial agent. The absence of growth does not necessarily mean that the antimicrobial agent has killed the microbes (bactericidal). It could mean that the antimicrobial agent has simply halted microbial cell division (bacteriostatic) . This discrepancy is resolved in the second microdialysis step, during which growth medium in the reaction chambers 100.3 is replaced with a drug-free medium. Resurgence of microbial growth under reagent-free culture implies that the antimicrobial agent applied during the first microdialysis step is bacteriostatic since it failed to eradicate or kill the microbes. Conversely, the absence of growth recovery implies that the antimicrobial agent was bactericidal, given that it rendered the microbes nonviable.

[0076] The second application of the microdialyser referred to above, is that of investigating confinement-induced drug tolerance (a possible underlying mechanism for macrophage-induced drug tolerance) and mycobacterial growth in the macrophage.

[0077] Persistence in mycobacteria has conventionally been investigated in large-volume contexts— such as test-tube liquid cultures or solid agar plates. Drug-tolerant persister cells routinely emerge in stationary phase cultures and biofilms where bacteria exist in high cell densities. Invariably, potentially drug-tolerant cells inoculated into drug-containing medium in vitro may fail to grow, not necessarily due to the potency of the drugs, but because the low microbial density at inoculation cannot permit mechanisms such as quorum sensing-mediated gene regulatory responses required for engendering the drug-tolerance phenotype. In this way, a drug formulation that is declared potent in vitro may fail to sterilise mycobacteria in in vivo contexts that may allow quorum-sensing- mediated drug-tolerance response— such as in high density biofilms or within macrophages.

[0078] The microdialyser of the invention provides a means of modelling persistence in mycobacteria in a volume-constrained growth environment. To begin with, the microscale reaction chambers 100.3 of the microdialyser are fabricated in a plurality of sizes based on the approximately 5 picoliter (0.005nL) volume of the membrane-bound compartment of the average human alveolar macrophage. In the embodiments of the invention described in this specification, the reaction chambers 100.3 are fabricated in the following sizes: 0.22nL; 0.5nL; 1 .2nL; 1 .7nL.

[0079] In addition, the microscale reaction chambers 100.3 are capable of attaining cell densities that approach intra-macrophage densities. Upon inoculation with between 1 and 5 mycobacteria in tests conducted by the applicant, the cell density in each microdialyser reaction chamber 100.3 was found to range from 10⁶ to 10⁸ cells/ml, thereby approximating the cell density (~10⁸ cells/ml) and physical confinement of intra- macrophage mycobacteria. The maximum cell densities attained in the microdialyser reaction chambers 100.3 (after culturing for several hours) routinely exceeded the maximum documented cell density for planktonic bacteria (10⁹ cells/ml) by at least two orders of magnitude. In this regard, microbial cell growth within the reaction chambers 100.3 may also mimic biofilm growth which, preliminary evidence suggests, could be an in vivo lifestyle of drug-resistant bacteria.

[0080] Besides the physical structure of the reaction chambers, the fluid exchange capabilities of the microdialyser approximate in vivo diffusive medium exchange. The arrangement of the conditioning chambers 102.3 and interconnection with reaction chambers 100.3 by way of diffusion channels 104.3 provide rapid, non-turbulent and non-invasive addition and removal of drugs, compounds and growth factors from the captive population of bacteria in the reaction chambers 100.3. This contrasts with conventional approaches used in medium exchange, which include chemostats and cell washing through centrifugation, which are chemically and physically disruptive techniques. Since the reaction chamber chambers 100.3 are arranged in parallel arrays, the microdialyser of the invention can support different, independent microbial cultures simultaneously that are independently accessible.

[0081 ] The global tuberculosis epidemic is largely attributable to the ability of Mycobacterium tuberculosis cells growing in human tissues to survive prolonged exposure to drugs, which, when tested outside the body, rapidly inhibit genetically identical cells. Alveolar macrophages have been implicated in inducing epigenetic mechanisms for drug tolerance by intra-macrophage mycobacteria, which make up the bulk of tuberculosis infection. Mycobacteria replicating in microdialysers have been found to survive exposure to drugs that inhibited genetically identical cells growing in conventional culture vessels. This suggests that volume-constrained or space-confined growth might be sufficient to induce drug tolerance in mycobacteria independently of macrophage- specific mechanisms.

[0082] While the microdialyser of the invention may not duplicate macrophage cell physiology, the device does allow some analysis of how some features of the in vivo growth environment may affect the drug- tolerance phenotype in mycobacteria. For instance, since the volume of the reaction chambers 100.3 can be selected (and adjusted with re- fabrication of the microdialyser), the fluidic confinement and physical dimensions of the micro-sized reaction chambers 100.3 constituted by the reaction chambers 100.3 can approximate the intracellular membrane- bound compartment of a macrophage or a granulomatous lesion of comparable size. Having an array of reaction chambers with differing internal volumes in the same device provides an opportunity to undertake simultaneous, parallel chemical analyses of a multiplicity of mycobacterial cell cultures to determine not only the reaction of the cell cultures to different drug cocktails, but also whether reaction chamber volume constraints affect the reaction of the cell cultures to the drugs. To some extent therefore, the microdialyser serves as a cell culture model for mycobacterial growth in the macrophage and demonstrates the use of the microdialyser to investigate macrophage-induced drug tolerance.

[0083] In practice, this implementation of the microdialyser could be used to identify drugs and drug cocktails best suited to overcome space- confined (which may include macrophage-induced) drug tolerance in clinical diagnostics.

[0084] To test the ability of the microdialyser to analyse complex drug susceptibility dynamics, the applicant cultured Mycobacterium smegmatis an experimentally tractable model organism for Mycobacterium tuberculosis in medium containing 350Mg/ml of rifampicin— a frontline antibiotic for the treatment of TB, using AFtsEX, a rifampicin-hypersensitive clone of Mycobacterium smegmatis, which has a minimum inhibitory concentration of 1 pg/ml for rifampicin. AFtsEX cells were cultured in the microdialyser chip within reaction chambers 100.3 of 4 different volumes: 0.22nL, 0.5nL, 1 .2nL and 1 .7nL. The mycobacteria exhibited positive growth in drug free medium and filled the reaction chambers 100.3 after 50 hours of growth, regardless of reaction chamber size. Remarkably, however, the growth rate varied as a function of reaction chamber volume, increasing with reaction chamber volume.

[0085] The applicant then cultured the microbes in microdialyser reaction chambers 100.3 in medium containing drug-free medium for 1 5 hours, which was replaced with medium containing rifampicin (350Mg/ml) for 1 50 hours and was then switched back to drug-free medium for 140 hours. During the first 1 5 hours of drug-free growth, all reaction chambers 100.3 registered positive growth regardless of reaction chamber volume. During the rifampicin regime: growth was inhibited in the bigger-volume reaction chambers 100.3 (1 .7nL— all 8 reaction chambers; 1 .2nL— all 8 reaction chambers; I 0.5nL— all 7 reaction chambers except one of the 0.5nL cultures, which continued to grow albeit at a slow-rate relative to the drug-free growth rate; surprisingly, growth was maintained in 5 of the 0.22nL reaction chambers 100.3 in the presence of rifampicin at a rate that was only slightly less than the drug-free growth rate.

Once the drug-containing medium was removed, all the reaction chambers 100.3 that had stopped growing during the rifampicin regime recovered.

[0086] These data suggest that in micro-size volume culture environments, rifampicin is bacteriostatic even at very high concentrations of the drug. In addition, growth-inhibition of AFtsEX microbes by rifampicin depended on reaction chamber volume, decreasing slightly in the smallest volume reaction chambers 0.22nL (relative to the drug-free equivalent) and disappearing altogether in the bigger reaction chambers 100.3.

[0087] Although the rifampicin-tolerance phenotype observed in the 0.22nL cultures was routinely reproduced in another strain of Mycobacterium smegmatis, it was absent in E. coll cells, suggesting its specificity to Mycobacterium smegmatis in this context. Whereas rifampicin suppressed growth in all the £ coli cultures, it was bactericidal in 83% of the reaction chambers 100.3 and bacteriostatic 1 7% of the reaction chambers 100.3 tested. Furthermore, microdialyser experiments with E. coli confirmed that rifampicin does retain its antibiotic potency in the microdialyser chip for over 200 hours. By contrast, in conventional drug susceptibility tests, AFtsEX microbes were susceptible to rifampicin at 35Mg/ml— this is 10 times less than the concentration that the microbes were resistant to in the microdialyser. [0088] Exactly why growth in the 0.22nL Mycobacterium smegmatis micro-cultures was maintained in rifampicin-containing medium and not the bigger reaction chambers 100.3 (0.5, 1 .2 and 1 .7nL) is unclear. Researchers have long observed that at high cell density (>10⁷ cells/ml), bacteria communicate to elicit complex patterns of co-operative behaviours that are advantageous for survival, behavior that is unattainable at low cell density and now generically termed "quorum sensing'". Documented responses include, among others, collective defense against competitive microorganisms, scavenging for nutrients, immune suppression and biofilm formation. Current models have shown that quorum sensitive bacteria secrete chemical signal molecules — autoinducers — whose concentration increases with cell density and reaching a critical threshold at which quorum sensing behaviour is triggered within the local cell population. Whereas quorum sensing is typically associated with large populations of bacteria, recent research findings suggest that even a single microbe confined within a sufficiently small growth chamber experiences a cell density that is high enough to elicit quorum sensing behaviour. Therefore, whereas mycobacteria lack the canonical quorum-sensing systems described in other bacteria, it is plausible that the drug-tolerance benefit that is exclusive to the smallest volume microdialyser reaction chambers 100.3 is a mediated by a non- canonical quorum-sensing system.

[0089] In addition to rifampicin, the applicant tested the susceptibility of /VI. smegmatis to other antibiotics used in the treatment of M. tuberculosis including ofloxacin and isoniazid. Microdialyser experiments showed that: ofloxacin (100Mg/ml) was bactericidal to /VI. smegmatis cells irrespective of the reaction chamber size; isoniazid was bactericidal regardless of reaction chamber size, provided that it was introduced to the microdialyser reaction chambers 100.3 when the cell density was low (less than 0.15 relative cell density units); conversely, when isoniazid was introduced to a reaction chamber that had a high cell density (greater than 0.15 relative cell density units), the drug lost its bactericidal effect on the microbes.

[0090] The microdialyser 300 illustrated in Figure 7 includes individually addressable reaction chambers 100.4 similar to those described above, arranged in modules of 20 each on either side of an array of cell loading ports 304 by means of which the reaction chambers 100.4 may be inoculated with mycobacterial cells. The microdialyser 300 includes a combinatorial mixer 302 that allows programmable combinatorial mixing of a plurality of reagent formulations, such as stock drug solutions mixed in various combinations. The reagents are introduced through external reagent inlet lines connected to reagent inlet ports 306 formed in the microdialyser 300. Arbitrary or predetermined combinations of the reagents (drugs or antimicrobial adjuvants such as efflux pump inhibitors) in variable concentrations can be pre-programmed using conventional or dedicated programs and dynamically mixed on-chip by the combinatorial mixer 302. By controlling the combinatorial mixer 302, any or all of the reaction chambers 100.4 may be charged with similar or different reagent mixtures. Control formulations that do not contain any reagent can also be included.

[0091 ] External control lines may be connected to control ports 308 that provide for membrane valve control on the microdialyser 300. Using the combinatorial mixer 302 as an example, the mixer 302 has 8 reagent inputs 306 (for instance reagents A to G) and the combinatorial mixer 302 recombines those inputs into arbitrary combinations at arbitrary minimum inhibitory concentrations (MICs), for example: Al O%[MIC] + B50%[MIC] + Cl00%[MIC] + DlO%[MIC] + ElO%[MIC] + FlO%[MIC] + + G20%[MIC]

[0092] Each unique formulation can then be targeted towards a sub- population of cells confined in a downstream chamber 100.4. The effect of each formulation on its target cell population can be ascertained via live-dead staining assay or direct imaging through microscopy.

[0093] The applicant constructed microdialysers according to the invention and conducted experiments to test the operation and efficacy of the microdialysers. Details of these microdialysers and experiments are described in a research article: Confinement-Induced Drug-Tolerance in Mycobacteria Mediated by an Efflux Mechanism, Luthuli BB, Purdy GE, Balagadde FK, PLoS ONE 10(8): eOl 36231— published: 21 August 2015. ¹ The entire article is incorporated herein by reference.

Claims

1 . A microfluidic device comprising a fluid flow circuit that includes an array of microfluidic chemical reaction chambers with differing internal volumes, the reaction chambers being configured to receive a chemical-containing fluid by way of the fluid flow circuit and to retain the fluid for the duration of all or part of a chemical reaction involving one or more of the chemicals contained in the fluid.

2. A microfluidic chemical assay system including the microfluidic device of claim 1 in which the microfluidic device reaction chambers are configured to permit simultaneous, parallel chemical analyses of an externally supplied analyte constituted by the chemical-containing fluid, the fluid flow circuit of the microfluidic device being configured to permit the introduction of the analyte to one or more predetermined reaction chambers.

3. The microfluidic chemical assay system of claim 2 in which the microfluidic reaction chambers of the microfluidic device are configured to form part of an analyte fluid flow circuit by means of which the analyte may be introduced into all or some of the microfluidic reaction chambers, the microfluidic device including a separate reagent fluid flow circuit that is configured to permit the introduction of an externally supplied reagent into all or some of the microfluidic reaction chambers.

4. The microfluidic chemical assay system of claim 3 in which the analyte circuit includes flow channels that are in fluid flow communication with valve-controlled individually controllable reaction chambers and the reagent flow circuit includes flow channels in fluid flow communication with a plurality of valve- controlled individually controllable microfluidic reagent chambers that are in valve-controlled fluid flow communication with the reaction chambers, the reagent circuit being configured to supply the reagent first to the reagent chambers and then to the reaction chambers.

5. The microfluidic chemical assay system of claim 4 in which valve control is provided by the inclusion of a control fluid circuit including a plurality of individually controllable valves configured to open and close any one or more flow channels, reaction chambers and reagent chambers.

6. A microfluidic bioassay system including the microfluidic device of claim 1 configured to analyse volume confinement behaviours in an analyte, the reaction chambers of the microfluidic device being dimensioned to have volumes ranging from 0.005nL†o 2nL.

7. The microfluidic bioassay system of claim 6 in which the reaction chambers are configured in a range of sizes from 0.22nL to 1 .7nL.

8. A microfluidic bioassay system including the microfluidic device of claim 1 in which the fluid exchange characteristics of the microfluidic device are configured to approximate in vivo diffusive medium exchange, the fluid flow channels between the microfluidic reagent and reaction chambers being dimensioned to restrict fluid exchange between the reagent and reaction chambers to passive diffusive exchange.

9. A method of modelling persistence in a microbial analyte in a volume-constrained growth environment using the microfluidic device of claim 1 , the method including the steps of: inoculating one or more reaction chambers of the microfluidic device with the microbial analyte; charging the inoculated reaction chamber/s with a growth medium within which to culture the microbial analyte mixed with a reagent constituted by an antimicrobial agent; and culturing the microbial analyte in the growth medium and recording growth or the absence of growth of the microbial analyte in the reaction chamber/s and, in particular, variations in growth rate, if any, as a function of reaction chamber volume.

10. A method of modelling persistence in a microbial analyte in a volume-constrained growth environment using the microfluidic device of claim 1 , the method including the steps of: inoculating one or more reaction chambers of the microfluidic device with the microbial analyte; charging the inoculated reaction chamber/s with growth medium free of antimicrobial agents within which to culture the microbial analyte; culturing the microbial analyte in the growth medium; recording growth or the absence of growth of the microbial analyte and variations in growth rate, if any, as a function of reaction chamber volume; replacing the drug-free growth medium in the inoculated reaction chamber/s with a similar growth medium including an antimicrobial agent; and recording growth or the absence of growth of the microbial analyte and variations in growth rate, if any, as a function of reaction chamber volume.

1 1 . The method of claim 10 including the additional steps of: replacing the drug-containing growth medium in the inoculated reaction chamber/s with drug-free growth medium; and recording growth or the absence of growth of the microbial analyte and variations in growth rate, if any, as a function of reaction chamber volume.

12. A method of resolving the bacteriostatic and bactericidal credentials of an antimicrobial agent using the microfluidic device of claim 1 , the method including the steps of: inoculating one or more reaction chambers of the microfluidic device with a microbial analyte; charging the inoculated reaction chamber/s with a growth medium within which to culture the microbial analyte mixed with a reagent constituted by an antimicrobial agent; culturing the microbial analyte in the growth medium and observing the progress of microbial growth, if any, in the reaction chamber/s; recording growth or the absence of growth of the microbial analyte in the reaction chamber/s and designating the microbial analyte in any chamber/s showing growth of the microbial analyte as being resistant to the antimicrobial agent; replacing the inoculated reaction chamber/s with a similar growth medium excluding the antimicrobial agent; recording the resurgence, if any, of growth of the microbial analyte in the reaction chamber/s; and in a final record, recording the antimicrobial agent as bacteriostatic in respect of the microbial analyte in any chamber/s showing resurgent growth and bactericidal in respect of the microbial analyte in any chamber/s in which growth recovery is not observed.