WO2024156586A1

WO2024156586A1 - A device, a system and a method for performing antibiotic susceptibility tests

Info

Publication number: WO2024156586A1
Application number: PCT/EP2024/051148
Authority: WO
Inventors: Zhen Zhang
Original assignee: Individual
Current assignee: Individual
Priority date: 2023-01-23
Filing date: 2024-01-18
Publication date: 2024-08-02
Anticipated expiration: 2025-07-23
Also published as: CN120641552A; EP4655385A1

Abstract

The disclosure relates to a micro chamber device (1) for performing antibiotic susceptibility tests on bacterial cells (12) of a range of bacterial cell sizes, the micro chamber device (1) being arranged on a substrate (40) The micro chamber device (1) comprising a micro chamber (11), a fluid inlet (14) and a fluid outlet (16) in fluid communication with the micro chamber (11), and an integrated sensor (18) arranged in the micro chamber (11), the sensor (18) being configured to monitor a property of bacterial cell activity in the micro-chamber (11) in real-time, and characterized in that the micro chamber (11) comprises at least one trapping device (20) configured to capture at least one bacterial cell (12) in the micro chamber (11). The disclosure also relates to a test system (2) comprising at least one micro chamber device (1) and to a method for performing antibiotic susceptibility tests on bacterial cells (12).

Description

A DEVICE, A SYSTEM AND A METHOD FOR PERFORMING ANTIBIOTIC SUSCEPTIBILITY TESTS

TECHNICAL FIELD

The present disclosure relates to a micro chamber device for performing antibiotic susceptibility tests on bacterial cells. In particular, it relates to a micro chamber configured to trap at least one bacterial cell of a predetermined range of bacterial cell sizes. The present disclosure also relates to a test system comprising at least one such micro chamber and to a method for performing antibiotic susceptibility tests on bacterial cells in the test system.

BACKGROUND

Rapid and precise treatment of infectious diseases is crucial to avoid costly and possibly even fatal complications resulting in increased morbidity of a patient, side effects of drugs, etc.

Antibiotic Susceptibility Tests (AST) prior to introduction of drug treatment can make sure a correct selection of antibiotics for precise treatment of the patient. It can also prevent the development of antibiotic resistance caused by abuse of the latest version of drugs.

A common way for AST is to use bacteria/cell growth or behaviour as an indicator for the presence of different antibiotics, such as classic phenotypic antibiotic resistance profiling methodology preformed via disk diffusion tests.

However, traditional methods of examining cell growth into colonies in culturing media in the presence of different drugs usually take more than two days. They often fail to guide precise medical treatment in the early, often critical, stages of disease development.

In addition, for many infectious diseases, retrieving bacterial samples of adequate bacterial amounts for the tests is also a major challenge. This usually demands a precultivation step for at least one day before starting the AST.

Furthermore, these conventional methods are based on detection of group behaviour of bacterial growth. They have limitations on samples containing sub-populations of slow- or non-growing bacteria, which can resume growth and cause a relapse of the infection after the antibiotic is removed. To be able investigate the impacts of these slower non-growing bacteria to infection treatments, new devices and methods for performing ASTs at single cell level are required. SUMMARY

Therefore, an object of the disclosure is to provide an improved device for antibiotic susceptibility test. More specifically, an object of the disclosure is to provide a micro chamber, a system and a method for more accurate tests and shortened testing time of antibiotics on single or multiple bacterial cells.

According to a first aspect of the present disclosure, the object is at least partly achieved by a micro chamber device according to claim 1.

Hence, there is provided a micro chamber device for performing antibiotic susceptibility tests on bacterial cells of a predetermined range of bacterial cell sizes. The micro chamber device is arranged on a substrate which extends laterally in a plane comprising a second axis and a third axis orthogonal to the second axis, the plane being orthogonal to a first axis. The micro chamber device comprises a fluid inlet and a fluid outlet in fluid communication with the micro chamber, and an integrated sensor arranged in the micro chamber, the sensor being configured to measure a property of bacterial cell activity in the micro chamber. The micro chamber comprises at least one trapping device configured to capture at least one bacterial cell in the micro chamber. The sensor is a field effect transistor-based ion sensor or a piezoresistive sensor.

The micro chamber may be provided by conventional semiconductor manufacturing technology at low cost in conventional semiconductor chip foundries. The micro chamber may thus be provided on a glass or semiconductor substrate or chip. The micro chamber is arranged for high throughput bacteria-collecting microfluidics for multiplexed rapid antibiotic susceptibility tests. The term multiplexed is herein to be understood as allowing simultaneous tests of different antibiotics in different micro chambers of a test system comprising the micro chamber devices. The small size of semiconductor devices may facilitate direct testing of bacterial sample without any need for pre-cultivation.

For reference, different types of bacterial cells vary in size, from a few hundred nanometres to approximately ten micrometres. Since the micro chambers are cheap to manufacture, the disclosed test systems may be manufactured and stored by a laboratory in different sizes intended for different ranges of bacterial cell diameters. Thus, when a drug is to be tested on a predetermined bacterial cell type, the size of the bacterial cell type may be determined, and a correspondingly dimensioned test system having micro chambers dimensioned for the relevant range of bacterial cell sizes may be selected from the stored test systems. Especially, a largest and a smallest cell extension of the predetermined bacterial cell type is determined. The relevant range is herein to be understood as the range of cell sizes including the determined cell size. Alternatively, if the bacterial cell type is not known, a test system comprising differently dimensioned micro chambers, where different micro chambers are dimensioned for different predetermined ranges of cell sizes, may be selected. Thereby, time is saved in that the bacterial cells do not need to be studied before the antibiotic susceptibility testing.

A predetermined range of cell sizes is herein to be understood as a range of cell sizes which allow isolation/detection of a limited range of bacterial cells/sizes, i.e. similarly sized, but not necessarily identically sized cells. Thereby, a particular micro chamber will be designed to accommodate cells of a limited range of sizes for testing. Other micro chambers may simultaneously be designed and used for other predetermined ranges of bacterial cell sizes, thereby allowing blind tests on cells of unknown types and sizes, since some micro chambers in a test system of micro chambers will be correctly dimensioned for the unknown cell type.

That the at least one bacterial cell is “captured” in the micro chamber is to be understood as the bacterial cell being either accommodated by the trapping device or loosely held by the trapping device or prevented from leaving the micro chamber by the trapping device. Loosely held is herein to be understood as the bacterial cell being able to move in its position, and at the same time being prevented from leaving the trapping device.

The fluid inlet is arranged to introduce a fluid into the micro chamber. The fluid may be a fluid containing a bacterial sample, i.e. bacterial cells or a fluid comprising culturing media for nourishing bacterial cells. Fluid comprising culturing media may, or may not, comprise antibiotics for performing tests on the bacterial cells.

Fluid introduced into the micro chamber may be evacuated through the fluid outlet. Before tests are performed on a bacterial sample in the micro chamber, the fluid inlet and/or the fluid outlet may be closed by a sealing fluid, such as oil, or by closing a valve with which fluid inlet and the fluid outlet may be in fluid communication.

The trapping device according to the disclosure may be configured in different ways to trap a single bacterial cell or a plurality of bacterial cells in the micro chamber. A trapping device may be configured to only capture, a single cell. An alternatively configured trapping device may capture multiple cells in the micro chamber. However, multiple single-cell trapping devices may also be arranged in the micro chamber if tests are to be performed on multiple bacterial cells. Thereby, it may be possible to study behaviour of single bacterial cells in some micro chambers versus multiple bacterial cells in other micro chambers of a test system comprising the micro chamber devices of the present disclosure.

The sensor may be an integrated nanoscale semiconductor electronic sensor for real time monitoring of bacterial activity. As such the sensor may be a field effect transistor- based ion sensor, such as a potentiometric ion sensor, which is configured to monitor bacterial cell metabolism by measuring metabolism-induced acidification of the culturing media. Alternatively, the sensor may be an integrated piezoresistive sensor which may monitor bacterial cell motion via the piezoresistive effect, such as of silicon, Si. Introduction of an effective drug (antibiotic) may inactivate the bacterial cells. From a sensing point of view, potentiometric sensing of pH using a field effect transistor is much more reliable than directly measuring a resistance (or resistivity) of the of the culturing media, since a contribution from a solid-liquid interface between electrodes/liquid may generate large and random errors to resistance measurements. In addition, directly monitoring changes in physiological parameters (such as pH of the culturing media or motion) can potentially be much faster than optically monitoring cell growth, since it does not rely on a visible morphology change for signal generation. Monitoring of physiological parameters can potentially profile the drug response of bacteria in real-time, using highly sensitive and reliable field effect transistor-based ion sensors or piezoresistive sensors. The effect of the drug may immediately be detected from reduced electrical signals generated by the cell metabolism or cell movement. The antibiotic susceptibility test is all-electrical and based on current-voltage measurements using conventional equipment. The sensor being “integrated” means that it may be manufactured by conventional semiconductor processing technology.

Herein is disclosed trapping and drug testing on single bacterial cells. As described in the background section above, some cell populations comprise sub-populations of slow, or non-growing cells. These cells are missed when performing conventional antibiotic tests on groups of cells. The herein-described single-cell trapping and testing allows study of single cell behaviour, and thereby also study of behaviour of such slow or non-growing cells.

Optionally, the micro chamber, the fluid inlet and the fluid outlet are formed of a single material on the substrate. Any microstructures of the present disclosure, such as walls and top cover of the micro chamber and any connecting channels, e.g. inlets and outlets, may be fabricated by conventional techniques using polydimethylsiloxane (PDMS), or glass, on a semiconductor or glass substrate. Thereby, manufacturing is greatly simplified.

Optionally, a lateral width along the third axis (y) of the micro chamber exceeds a plurality of bacterial cells as seen along a largest cell extension (c) of the largest bacterial cell size of the predetermined range of bacterial cell sizes, and wherein the at least one trapping device is configured to accommodate a single bacterial cell, and wherein the at least one trapping device is arranged in the micro chamber between the fluid inlet and the fluid outlet.

In one example, the lateral width of the micro chamber allows bacterial cells to flow with the fluid comprising the bacterial cells, from the fluid inlet to the fluid outlet. At least one trapping device is arranged between the fluid inlet and the fluid outlet and is configured to accommodate/capture a single bacterial cell. As such, the trapping device substantially corresponds to the bacterial cell in size. In other words, the trapping device is large enough to capture the bacterial cell, but when one bacterial cell has been captured by the trapping device, the trapping device is full, i.e. occupied and may not accommodate any more bacterial cells.

Optionally, the fluid inlet and the fluid outlet are arranged on laterally opposite sides of the at least one trapping device. Thereby, the at least one trapping device is arranged in the flow of the fluid comprising the bacterial sample.

Optionally, the trapping device is a structure having a lateral width along the third axis and a height along the first axis corresponding to the largest cell extension of the predetermined range of bacterial cell sizes. The lateral extension and height are thus configured to match the largest extension of a bacterial cell. The trapping device of this example may be a structure arranged on the substrate. The structure may be formed of the same single material as the micro chamber, the fluid inlet and the fluid outlet.

Optionally, the trapping device has a concave surface facing the fluid inlet, the concave surface being arranged to accommodate a single bacterial cell and wherein the trapping device further has a fluid channel extending through the trapping device in a direction from the concave surface towards the fluid outlet for directing a bacterial cell towards the trapping device to be accommodated by the concave surface.

The concave surface may thus act as a bowl in which a single bacterial cell may be collected and accommodated. The fluid channel extending through the trapping device further provides a flow of the fluid, through the trapping device which guides bacterial cells of a liquid bacterial sample flowing into the micro chamber to the concave bowl-shaped surface of the trapping device. The fluid channel may be configured small enough to prevent bacterial cells from entering the fluid channel, and large enough to allow fluid flow therethrough.

Optionally, the micro chamber further comprises a drain trench for evacuating fluid from the micro chamber, the drain trench extending along the first axis from the micro chamber through the substrate, and wherein the trapping device comprises nanowires extending laterally across the micro chamber, such that the fluid inlet and the fluid outlet are on an opposite side of the nanowires in relation to the drain trench, the nanowires further comprising the at least one trapping device, in the form of a nanowire net having a lateral extension along the second axis and the third axis, which lateral extension corresponds to a largest cell extension of the predetermined range of bacterial cell sizes to accommodate a single bacterial cell at the trapping device.

Thereby, during loading of bacterial cells in the micro chamber, fluid comprising the bacterial sample/cells is introduced into the micro chamber via the fluid inlet and evacuated via the fluid outlet. After a predetermined time period, the micro chamber is loaded. The fluid outlet may thereafter be closed. The micro chamber may be provided on a substrate. The drain trench may be etched through the substrate and may be fluidly connected to tubing, which tubing may comprise an openable valve. After loading of the micro chamber and closing of the fluid outlet, culturing media may be introduced into the micro chamber via the fluid inlet and evacuated via the drain trench. When flowing culturing media through the micro chamber, a single bacterial cell will be captured/stopped on the trapping device/nanowire net, while the rest of bacterial cells in the micro chamber will be washed out via the drain trench.

The nanowires and the nanowire net are understood to be formed by conventional semiconductor processing technology in conjunction with forming the drain trench through the substrate. The nanowires and nanowire net are thus integrated and monolithic components with the substrate. The nanowires and the net of nanowires may have a cross- sectional diameter ranging between approximately five nanometers and a few hundred nanometers.

Optionally, the trapping device is surface-functionalized to cause loose attachment of the bacterial cell to the trapping device. The trapping device may be surface- functionalized by conventional means, such as by a stable monolayer of organic molecules ((3-aminopropyl)triethoxysilane (APTES). The functionalization makes the nanowire net biocompatible and improves electronic cell-sensor coupling. The loose attachment of the bacterial cell allows the bacterial cell to move in its position but prevents the bacterial cell from leaving the trapping device.

The trapping device may in this example comprise the sensor. The sensor may be a piezoelectric sensor. As such movement of the bacterial cell on the nanowire net causes vibrations in the nanowire net/trapping device, which vibrations electrical signals that may be measured to study bacterial motion.

Optionally, the fluid inlet comprises a plurality of first openings having a first lateral width configured to allow a predetermined range of bacterial cell sizes or smaller objects to pass through the first openings, and wherein the fluid outlet comprises a plurality of second openings of a second lateral width configured to prevent bacterial cells of the predetermined range of bacterial cell sizes or larger objects from passing through the second openings.

The plurality of first openings enables filtering of large particles or cells and allows a rapid inflow of fluid via the fluid inlet into the micro chamber. The plurality of second openings further enables cells of the predetermined range of cell sizes to be kept in the micro chamber and allows a rapid outflow of fluid from the micro chamber via the fluid outlet.

Optionally, the trapping device comprises the first openings and the second openings. The first openings prevent larger particles from entering the micro chamber. The second openings may act as a filter to prevent the bacterial cells of the fluid comprising the bacterial sample from leaving the micro chamber. Thereby, the first openings and second openings act as the trapping device for capturing multiple bacterial cells in the micro chamber.

According to a second aspect of the present disclosure, the object is at least partly achieved by a test system according to claim 11 .

Hence there is provided a test system for performing antibiotic susceptibility tests on at least one bacterial cell, the test system comprising at least one micro chamber device according to any one of the embodiments of the first aspect of the disclosure, the test system further comprising at least a first channel fluidly connected to the fluid inlet of the at least one micro chamber for supplying a liquid to the micro chamber, at least a second channel fluidly connected to the fluid outlet of the at least one micro chamber for evacuating the liquid from the micro chamber.

Optionally, the test system comprises a plurality of micro chamber devices, wherein at least some of the plurality of micro chamber devices are differently dimensioned for bacterial cells of different predetermined ranges of bacterial cell sizes.

The test system may thus comprise differently dimensioned micro chambers, where different micro chambers are dimensioned for respective predetermined ranges of cell sizes, which allows so-called blind testing on unknown bacterial cell types whose dimensions have not yet been determined. Blind testing may save time since the bacterial cells do not have to be identified or measured to learn which antibiotic is effective against the cells.

The test system may comprise at least one array of micro chambers interconnected by first and second channels. Each array of micro chambers may be interconnected by respective first and second channels. Each array of micro chambers may, during testing, be supplied with different culturing media, i.e. with or without antibiotics or with different antibiotics to enable multiplexed testing of antibiotics. The fluid inlets and the fluid outlets of the micro chambers comprised in the test system may be closed by closing the first channel and the second channel. The fluid inlets and the fluid outlets may be closed by closing valves to which the first channel and the second channel are connected. Alternatively, the fluid inlets and the fluid outlets may be closed by filling the first channel and the second channel with a sealing fluid, such as oil. The sealing fluid may provide electrical insulation between micro chambers to improve accuracy and reliability of sensor measurements.

Optionally, the test system further comprises a monitoring unit for monitoring bacterial cell activity in the at least one micro chamber, the monitoring unit being electrically connected to the sensor of the at least one micro chamber.

The monitoring unit may be electrically connected to the test system to receive signals from each of the sensors of the micro chambers comprised in the system. The monitoring unit may interpret measurements of the sensors and provide a rapid presentation of the bacterial cells’ response to the antibiotics supplied to the micro chambers.

According to a third aspect of the present disclosure, the object is at least partly achieved by a method according to claim 13.

Hence there is provided a method for performing antibiotic susceptibility tests on at least one bacterial cell of at least one predetermined range of bacterial cell sizes in the test system according to any of the embodiments of the second aspect of the disclosure. The method comprises via the at least first channel, loading the at least one micro chamber with a liquid bacterial sample, capturing, by the trapping device, at least one bacterial cell in the at least one micro chamber, loading culturing media, with or without antibiotics, to each of the at least one micro chamber, and measuring, by the sensor, at least one property of bacterial cell activity of the captured cell in each of the at least one micro chamber.

Accordingly, the method allows antibiotic susceptibility testing on bacterial cells of unknown size, such as on an unknown bacterial cell type whose cell size has not yet been determined. This is achieved by using a test system comprising differently dimensioned micro chambers, where different micro chambers are dimensioned for different predetermined cell sizes. Micro chambers which are correctly dimensioned for the unknown cell type will provide relevant measurements from their respective sensors.

Loading of the bacterial sample is thus performed by supplying a liquid bacterial sample via the at least first channel and into the micro chamber via the fluid inlet. The liquid bacterial sample flows from the fluid inlet to the fluid outlet and into the at least second channel. During the predetermined time period it may, in some examples, be statistically ensured that at least one bacterial cell will be captured by the at least one trapping device of the micro chamber. Thereafter, culturing media/liquid is loaded into the at least one micro chamber for a predetermined time period, which culturing media may or may not comprise an antibiotic for testing on the at least one bacterial cell. During the predetermined time period it may, in some examples, be statistically ensured that at least one bacterial cell will be captured by the at least one trapping device of the micro chamber. The loading of culturing media will evacuate all, or most, bacterial cells not captured by the at least one trapping device, unless prevented from leaving the micro chamber by any second openings of the fluid outlet. The method may comprise stopping loading of culturing media before measuring cell activity of the at least one bacterial cell. In this manner, the electrical measurement by the sensor of the at least one bacterial cell activity may be performed in a controlled liquid environment.

Optionally, the method comprises closing the fluid inlet and/or the fluid outlet of the at least one the micro chamber device by closing the at least first channel and/or second channel. Thereby, control of the liquid environment in the at least one micro chamber is further improved.

In one example, the method comprises loading culturing media to each of the at least one micro chamber and evacuating the culturing media through the drain trench, during which process a bacterial cell will be captured/stopped by the nanowire net, and the rest of bacterial cells in the micro chamber 11 will be washed out via the drain trench.

Optionally, the properties measured by the sensor are metabolism-induced acidification of the culturing media and/or bacterial motion., which properties are easily measured by the herein disclosed sensors.

Optionally, the method may comprise evacuating the liquid bacterial sample via the drain trench. The fluid outlet may be closed by the sealing fluid and the liquid bacterial sample may be evacuated via the tubing which may be fluidly connected to the drain trench. The fluid outlet may alternatively be closed by closing a valve to which the second channel is connected.

The above aspects, accompanying claims, and/or examples disclosed herein above and later below may be suitably combined with each other as would be apparent to anyone of ordinary skill in the art.

Additional features and advantages are disclosed in the following description, claims, and drawings, and in part will be readily apparent therefrom to those skilled in the art or recognized by practicing the disclosure as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

Further objects and advantages of, and features of the disclosure will be apparent from the following description of one or more embodiments, with reference to the appended drawings, where:

Fig. 1 shows a conceptual top-down view of a micro chamber device according to a first aspect of the disclosure.

Fig. 2 shows an example of the conceptual micro chamber device of Fig. 1 comprising a plurality of single-cell trapping devices.

Fig. 3 shows a cross-sectional side view of the micro chamber device of Fig. 1 .

Fig. 4 shows a top-down view of a micro chamber device according to an example of the disclosure.

Fig. 5 shows a cross-sectional side view of the micro chamber device of Fig. 4.

Fig. 6 shows a top-down view of a micro chamber device according to an example of the disclosure.

Fig. 7 shows a cross-sectional side view of the micro chamber device of Fig. 6.

Fig. 8 shows a cross-sectional side view of the micro chamber device of Fig. 6.

Fig. 9 shows a top-down view of a micro chamber device according to an example of the disclosure.

Fig. 10 shows a cross-sectional side view of the micro chamber device of Fig. 9.

Fig. 11 shows a top-down view of an exemplary test system according to a second aspect of the disclosure.

Fig. 12 shows a cross-sectional side view of the test system of Fig. 11 .

Fig. 13 shows a top-down view of an exemplary test system according to a second aspect of the disclosure.

Fig. 14 shows a cross-sectional side view of the test system of Fig. 13.

Fig. 15 shows a top-down view of an exemplary test system according to the second aspect of the disclosure.

Fig. 16 shows a flowchart of a method according to a third aspect of the disclosure.

Fig. 17 shows a flowchart of a method according to an example of the third aspect of the disclosure. DETAILED DESCRIPTION OF EXAMPLE EMBODIMENTS OF THE INVENTION

The present disclosure is developed in more detail below referring to the appended drawings which show examples of embodiments. The disclosure should not be viewed as limited to the described examples of embodiments. Like numbers refer to like elements throughout the description.

The terminology used herein is for the purpose of describing particular aspects of the disclosure only and is not intended to limit the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Fig. 1 shows a conceptual top-down view of a micro chamber device 1 for performing antibiotic susceptibility tests on bacterial cells 12 of a predetermined cell size according to a first aspect of the disclosure. The micro chamber device 1 is arranged on a substrate 40 which extends in a plane A comprising comprising a second axis x and a third axis y orthogonal to the second axis x, the plane A being, orthogonal to a first axis z. The micro chamber device 1 comprises a micro chamber 11 , a fluid inlet 14, a fluid outlet 16 in fluid communication with the micro chamber 11 and a sensor 18 arranged in the micro chamber 11 . The sensor 18 is configured to measure a property of bacterial cell activity in the micro chamber 11. The micro chamber 11 further comprises at least one trapping device 20 configured to capture at least one bacterial cell 12 in the micro chamber 11 .

Any microstructures of the present disclosure, such as walls and top cover of the micro chamber 11 and any connecting channels, may be fabricated by conventional techniques of a single material using of instance polydimethylsiloxane (PDMS), or glass, on a semiconductor or glass substrate 40.

Fig. 1 is a conceptual illustration in that the trapping device 20 is illustrated as a box having a dashed outline. The trapping device 20 according to the disclosure may be configured in different ways to trap a single bacterial cell 12 or a plurality of bacterial cells 12 in the micro chamber. One trapping device 20 may be configured to only capture a single bacterial cell 12. An alternatively configured trapping device 20 may capture multiple bacterial cells 12 in the micro chamber 11. However, multiple single-cell trapping devices 20 may also be arranged in the micro chamber 11 , as shown in Fig. 2, such that each trapping device 20 captures one bacterial cell 12, e.g. if tests are to be performed on multiple bacterial cells. The illustrated dashed box only symbolizes the existence of a trapping device 20 in the micro chamber 11 .

Fig. 3 is a cross-sectional side view of the micro chamber of Fig. 1.

The arrows illustrate a flow of liquid into the micro chamber 11 via the fluid inlet 14, through the micro chamber 11 , to the fluid outlet 16. The liquid may for instance be a liquid bacterial sample comprising bacterial cells 12, or a culturing media comprising nutrition for bacterial cells. Culturing media may also be with or without a drug, or antibiotic, to be tested on the at least one bacterial cell 12 captured in the micro chamber 11 . The fluid inlet 14 and the fluid outlet 16 may comprise first openings and second openings, respectively, such that fluid inlet 14 and the fluid outlet 16 are restricted by the respective first and second openings.

Different types of bacterial cells 12 vary in size, from hundreds of nanometres to tens of micrometres. Since the micro chambers 11 are cheap to manufacture, disclosed test systems 2, comprising a plurality of micro chamber devices 1 , may be manufactured and stored by a laboratory in different sizes intended for different bacterial cell diameters. Thus, when a drug is to be tested on a predetermined bacterial cell type, the size of the bacterial cell type may be determined and a correspondingly dimensioned test system 2 (to be described below) may be selected from the stored test systems 2. for the antibiotic susceptibility tests. Especially, a largest cell extension and a smallest cell extension of the predetermined bacterial cell type is determined. Alternatively, if the bacterial cell type is not known, a test system 2 comprising differently dimensioned micro chambers 11 , where different micro chambers 11 are dimensioned for different predetermined cell sizes, may be selected. Thereby, time is saved in that the bacterial cells 12 do not need to be studied before the antibiotic susceptibility testing.

The sensor 18 may be a nanoscale semiconductor electronic sensor 18 for real time monitoring of bacterial cell activity. The sensor 18 may be integrated on the substrate. As such the sensor 18 may be a field effect transistor-based ion sensor 18 which is configured to monitor bacterial cell metabolism by measuring metabolism- induced acidification of the culturing media. Alternatively, the sensor 18 may be a piezoresistive sensor 18 which may monitor bacterial cell motion via the piezoresistive effect, such as of silicon (Si). Introduction of an effective drug (antibiotic) may inactivate the bacterial cells 12. The effect of the drug may be detected via reduced electrical signals generated by the bacterial cell metabolism/activity. The antibiotic susceptibility test is all-electrical and based on current-voltage measurements using conventional equipment. An example of a trapping device is illustrated in Fig. 4, which shows a top-down view of a micro chamber device 1. A lateral width W along the third axis y of the micro chamber 11 exceeds a plurality of bacterial cells, measured along a largest cell extension c of the largest bacterial cell size of the predetermined range of bacterial cell sizes. The at least one trapping device 20 is configured to accommodate a single bacterial cell 12. The at least trapping device 20 is arranged in the micro chamber 11 between the fluid inlet 14 and the fluid outlet 16.

The lateral width W of the micro chamber 11 allows bacterial cells 12 to flow with the fluid comprising the bacterial cells 12, e.g. the bacterial sample, from the fluid inlet 14 to the fluid outlet 16. At least one trapping device 20, or trapping device 20, is arranged between the fluid inlet 14 and the fluid outlet 16, each of the at least one trapping device 20 being configured to capture a single bacterial cell 12. As such, the trapping device 20 substantially corresponds to the bacterial cell 12 in size. In other words, the trapping device 20 is large enough to accommodate the bacterial cell 12, but when one bacterial cell 12 has been captured by the trapping device 20, the trapping device 20 is full/occupied and may not accommodate any more bacterial cells 12.

The fluid inlet 14 and the fluid outlet 16 are arranged on laterally opposite sides of the at least one trapping device 20. Thereby, the at least one trapping device 20 is arranged in the flow of the fluid comprising the bacterial sample during loading of the bacterial sample into the micro chamber 11 .

The trapping device 20 may be a structure having a lateral width w along the third axis y and a height h along the first axis z, shown in Fig. 5, configured to capture a single bacterial cell 12. The lateral width w and height h are thus configured to correspond to the largest extension of the largest cell size of the predetermined range of cell sizes.

The trapping device 20 may further have a concave surface 22 facing the fluid inlet, the concave surface 22 being arranged to accommodate a single bacterial cell 12. The trapping device 20 further has a fluid channel 24 extending through the trapping device in a direction from the concave surface 22 towards the fluid outlet 16 for directing a bacterial cell 12 towards the trapping device 20 to be accommodated by the concave surface 22. The concave surface 22 may thus act as a bowl in which a single bacterial cell 12 may be collected. The fluid channel 24 further provides a flow of the fluid through the trapping device 20 during use, which flow guides bacterial cells 12 towards the trapping device 20 to be accommodated/captured by the concave bowl-shaped surface 22 of the trapping device 20.

An alternative example of the trapping device 20 is shown in Fig. 6, which illustrates a micro chamber 11 comprising a drain trench 26 for evacuating fluid from the micro chamber 11 , the drain trench 26 extending along the first axis z from the micro chamber 11 through the substrate 40. The trapping device 20 comprises nanowires 28 extending laterally across the micro chamber 11 , such that the fluid inlet 14 and the fluid outlet 16 are on an opposite side of the nanowires 28 in relation to the drain trench 26, as seen along the first axis z. The nanowires 28 further comprise the at least one trapping device 20 in the form of a nanowire net 20 having a lateral extension along the second axis x and the third axis y which lateral extension corresponds to a largest cell size extension c of the predetermined range of bacterial cell sizes to accommodate a single bacterial cell 12 at the trapping device 20.

Also shown in Fig. 6 are a first channel 34 and a second channel 36 which deliver and collect fluid to and from the micro chamber 11 , respectively. The first channel 34 and the second channel 36 are comprised in a test system 2 according to a second aspect of the disclosure, to be described more in detail below.

During loading of a bacterial cells 12 in the micro chamber 11 , fluid comprising the bacterial sample/bacterial cells 12 is introduced into the micro chamber 11 via the fluid inlet 14 and evacuated via the fluid outlet 16, as shown in Fig. 7. When the micro chamber 11 is loaded, the fluid outlet 16 may be closed, as symbolized by the dashed area in Fig. 8. The fluid outlet 16 may be closed by closing a valve (not shown) to which the second channel 36 is connected. The drain trench 26 may be etched through the substrate 40 and may be fluidly connected to tubing (not shown) on an opposite side of the substrate 40 in relation to the micro chamber 11 . The dashed line 42 symbolizes an interface with the tubing and indicates that the drain trench 26 may be closed by a valve connected to the tubing (not shown) or by filling the tubing with the sealing fluid 32. After loading of the micro chamber 11 and closing of the fluid outlet 16, culturing media may be introduced into the micro chamber 11 via the fluid inlet 14 and evacuated via the drain trench 26 as shown by the arrows in Fig. 8, during which process a bacterial cell 12 will be captured/stopped by the nanowire net, and the rest of bacterial cells 12 in the micro chamber 11 will be washed out via the drain trench 26.

The nanowires 28 and the nanowire net/trapping device 20 are understood to be formed by conventional semiconductor processing technology in conjunction with forming the drain trench 26 through the substrate 40. The nanowires 28 and nanowire net/trapping device 20 are thus integrated and monolithic components with the substrate 40. The nanowires 28 and the trapping device 20 may have a cross-sectional diameter ranging between approximately five nanometers and a few hundred nanometers. The exemplary trapping device 20, exemplified by the nanowire net, may be surface- functionalized to cause loose attachment of a bacterial cell 12 to the nanowire net. The nanowire net 20 may be surface-functionalized by conventional means, such as by a stable monolayer of organic molecules ((3-aminopropyl)triethoxysilane (APTES). The functionalization makes the nanowire net biocompatible, and may improve electronic coupling between the bacterial cell 12 and the sensor 18.

The trapping device 20 in the form of the nanowire net may in this example comprise the sensor 18. The sensor 18 may be a piezoelectric sensor integrated in the nanowires 28. As such, movement of the captured bacterial cell 12 on the nanowire net/trapping device 20 causes vibrations in the nanowire net/trapping device 20, which vibrations result in electrical signals by the piezoelectric effect, which electrical signals may be measured by the sensor 18 to study bacterial motion.

In another example shown in Fig. 9, the fluid inlet 14 may comprise a plurality of first openings having a first lateral width D configured to allow bacterial cells 12 of the predetermined range of bacterial cell sizes or smaller to pass through the first openings. The fluid outlet 16 may further comprise a plurality of second openings of a second lateral width d configured to prevent bacterial cells 12 of the predetermined range of bacterial cell sizes or larger from passing through the second openings.

The plurality of first openings enables filtering of large particles or cells, and allows a rapid inflow of fluid via the fluid inlet 14 into the micro chamber 11. The plurality of second openings further enables bacterial cells 12 of the predetermined range of bacterial cell sizes or smaller to be kept inside the micro chamber 11 and allows a rapid outflow of fluid from the micro chamber 11 via the fluid outlet 16.

In this exemplary embodiment, the trapping device 20 comprises the first openings and the second openings. The trapping device 20 thus corresponds to the entire micro chamber device 1 , including first and second openings. The second openings may act as a filter to prevent the bacterial cells 12 of the fluid comprising the bacterial sample from leaving the micro chamber 11. Thereby, the first openings and second openings act to capture multiple bacterial cells in the micro chamber 11.

Fig. 10 is a side view of the micro chamber device of Fig. 9. As described hereinabove, in order to close the micro chamber 11 , the fluid inlet 14 and the fluid outlet 16 may be closed by closing valves (not shown) to which the first channel 34 and the second channel 36 are connected. Alternatively, the fluid inlet 14 and the fluid inlet 16 may be closed by filling the first channel and the second channel with a sealing fluid 32, such as oil, as symbolized by the dashed areas 32 in Fig. 10. A micro chamber 11 sealed by sealing fluid 32 may yield more accurate measurements by the sensor 18 since the micro chambers 11 may be electrically insulated from each other by the sealing fluid 32.

As shown in Figs 6-8, the first openings and the second openings may advantageously be employed as a filter in combination with the nanowire net trapping device 20. A plurality of bacterial cells 12 will thereby be captured in the micro chamber 11 during bacterial loading. During subsequent loading of culturing media a single bacterial cell 12 will be captured by the nanowire net trapping device 20, and the rest of the bacterial cells 12 in the micro chamber 11 will be washed out via the drain trench 26.

A second aspect of the disclosure is illustrated in Figs 11-13. Fig. 11 shows a test system 2 for performing antibiotic susceptibility tests on at least one bacterial cell 12 of at least one predetermined range of bacterial cell sizes. The test system 2 comprises at least one micro chamber device 1 according to any one of the embodiments of the first aspect of the disclosure. The test system 2 further comprises at least a first channel 34 fluidly connected to the fluid inlet 14 of the at least one micro chamber 11 for supplying a liquid to the micro chamber 11. The test system 2 further comprises at least a second channel 36 fluidly connected to the fluid outlet 16 of the at least one micro chamber 11 , for evacuating the liquid from the micro chamber 11.

The test system 2 may comprise a plurality of micro chamber devices 1 , wherein at least some of the plurality of micro chamber devices 1 are differently dimensioned in relation to each other, for performing test on bacterial cells 12 of different predetermined ranges of bacterial cell sizes.

The test system 2 may thus comprise differently dimensioned micro chambers 11 , where different micro chambers 11 are dimensioned for different predetermined ranges of bacterial cell sizes, which allows so-called blind testing on unknown bacterial cell types whose dimensions have not yet been determined. Blind testing may save time since the bacterial cells 12 do not have to be identified or measured to learn which antibiotic is effective against the cells.

The test system 2 may comprise at least one array 44 of micro chamber devices 1 interconnected by the at least first channel 34 and the second channel 36. Fig. 11 shows three micro chambers 11 of an array 44 of micro chamber devices 1. However, any number of micro chamber devices 1 may be comprised in the array. The array is defined as set of micro chamber devices 1 being interconnected by the same first channel 34 and the same second channel 34. The test system 2 may further comprise a plurality of arrays 44 of micro chamber devices, each array 44 of micro chamber devices 1 interconnected by respective first channels 34 and second channels 36. Each array 44 of micro chamber devices 1 may, during testing, be supplied with different culturing media, i.e. with or without antibiotics, or with different antibiotics. The fluid inlets 14 and the fluid outlets 16 of the micro chambers 11 comprised in the test system 2 may be closed by closing the first channel 34 and the second channel 36. The fluid inlets 14 and the fluid outlets 16 may be closed by closing valves (not shown) to which the first channel 34 and the second channel 36 are connected. Alternatively, the fluid inlets 14 and the fluid outlets 16 may be closed by filling the first channel 34 and the second channel 36 with a sealing fluid, such as oil.

It should be noted that the micro chamber devices 1 illustrated in Fig. 11 depict the conceptual micro chamber devices of Figs 1-3. The fluid inlet 14 and the fluid outlet 16 of the micro chamber 11 may therefore also comprise the first openings and second openings, respectively, illustrated in Fig. 9. The dashed box symbolizing the trapping device may thus either represent a structure protruding from the substrate 40 as shown in Fig. 4 and Fig. 5, or a nanowire net extending over a drain trench 26, as shown in Figs 6-8. The structure and the nanowire net are described hereinabove.

Fig. 12 shows an exemplary side view of the test system 2 of Fig. 11 , wherein liquid is shown to enter a micro chamber 11 from the first channel 34 and to exit the micro chamber 11 via the fluid outlet 16 to the second channel 36.

Fig. 13 and Fig. 14 show a test system 2 comprising micro chamber devices 1 as shown in Fig. 9 and Fig. 10. The trapping device 20 is herein to be understood as the whole micro chamber 11 , including the first openings and the second openings of the respective fluid inlet 14 and the fluid outlet 16. The trapping device is in this example a multi-cell trapping device, where a number for bacterial cells are captured in the micro chamber 11 due to the second openings of the fluid outlet 16, which openings are configured to be too narrow for bacterial cells 12 to leave the micro chamber 11. Testing and measuring is thereby performed on a group of bacterial cells 12.

Fig. 15 shows an example of three arrays 44 of micro chamber devices 1. Each array may be supplied with a different composition of culturing media during use. As such, one array may be supplied with antibiotics, whereas another array may be supplied with culturing media without antibiotics. Also, different antibiotics may be introduced to different arrays. Each array may further comprise differently dimensioned micro chamber devices 1 for performing tests on bacteria of unknown size, so-called blind tests.

The test system 2 may be provided on a substrate 40. The test system may be configured to be fluidly connected to a source of a liquid bacterial sample (not shown) and/or to a source of liquid culturing media (not shown). The test system 2 may further comprise a monitoring unit 38 electrically connected to the sensor 18 of the at least one micro chamber device 1. The monitoring unit 38 may be electrically connected to the sensors 18 of the test system 2 to receive signals from each of the sensors 18 of the micro chambers 11 comprised in the test system 2. The monitoring unit 38 may interpret measurements of the sensors 18 and provide a rapid presentation of the bacterial cells’ 12 response(s) to the antibiotics supplied to the micro chambers 1.

A third aspect of the disclosure is shown in Fig. 16., depicting a flowchart of a method 3 for performing antibiotic susceptibility tests on at least one bacterial cell 12 in the test system 2 according to any of the embodiments of the second aspect of the disclosure. Unless specified, actions S2, S3 are not carried out in any particular order in relation to each other. The method 3 comprises: via the at least first channel 34, loading S1 the at least one micro chamber 11 with a liquid bacterial sample comprising bacterial cells 12, capturing S2, by the trapping device 20, at least one bacterial cell 12 in the at least one micro chamber 11 , loading S3 culturing media, with or without antibiotics, to each of the at least one micro chamber 11 , and measuring S4, by the sensor 18, at least one property of bacterial cell activity of the captured bacterial cell 12 in each of the at least one micro chamber 11 .

Loading S1 of the bacterial sample is thus performed by supplying a liquid bacterial sample via the at least first channel 34 into the micro chamber 11 via the fluid inlet 14. The liquid bacterial sample flows from the fluid inlet 14 to the fluid outlet 16 and into the at least second channel 36 for a predetermined time period. In some examples, it is statistically ensured that at least one bacterial cell 12 will be captured by the at least one trapping device 20 of the micro chamber 11 during the predetermined time period . Subsequently, the liquid bacterial sample is evacuated by loading S3 culturing media/liquid into the at least one micro chamber 11. In some examples, a bacterial cell 12 is captured by the trapping device 20 during loading S3 of culturing media. The culturing media may or may not comprise an antibiotic for testing on the at least one bacterial cell 12. The method may comprise stopping S6 loading of culturing media. In this manner, the electrical measurement by the sensor 18 of the at least one bacterial cell activity may be improved by performing the measurement in a more controlled liquid environment.

The method 3 may comprise closing S5 the fluid inlet 14 and/or the fluid outlet 16 of the at least one micro chamber device 1 . The fluid inlet 14 and/or the fluid outlet 16 may be closed by closing valves (not shown) to which the at least first channel 34 and the at least second channel 36 are connected, or by filling the at least first channel 34 and/or second channel 36 with a sealing medium 32. Thereby, control of the liquid environment in the at least one micro chamber 11 is further improved. In one example, shown in Fig. 17, the action of loading S1 the micro chamber 11 with the liquid bacterial sample is followed by closing S5 the second channel 36 and loading S3 culturing media into each of the at least one micro chamber 11. The loading S3 of culturing media results in capturing/stopping S2 bacterial cells 12 on the nanowire net/trapping device 20 while simultaneously evacuating the culturing media through the drain trench 26 as shown by the arrows in Fig. 8. During loading S3 of culturing media a single bacterial cell will be captured by the nanowire net trapping device 20 due to the flow of culturing media being perpendicular to the lateral extension of the nanowire net/trapping device 20, and the rest of the bacterial cells 12 in the micro chamber 11 will be washed out via the drain trench 26. The example method 3 may, as described above, optionally comprise stopping S6 loading of culturing media before measuring S4 cell activity of the at least one bacterial cell. The first channel 34 and the second channel 36 may optionally be closed S5, as described above, before the measuring S4 of cell activity is started.

The property measured by the sensor 18 may be metabolism-induced acidification of the culturing media and/or bacterial motion.

Claims

1. A micro chamber device (1) for performing antibiotic susceptibility tests on bacterial cells (12) of a predetermined range of bacterial cell sizes, the micro chamber device (1 ) being arranged on a substrate (40) which extends laterally in a plane (A) comprising a second axis (x) and a third axis (y) orthogonal to the second axis (x), the plane (A) being orthogonal to a first axis (z), the micro chamber device (1) comprising a micro chamber (11), a fluid inlet (14) and a fluid outlet (16) in fluid communication with the micro chamber (11), and an integrated sensor (18) arranged in the micro chamber (11), the sensor (18) being configured to measure a property of bacterial cell activity in the micro chamber (11), and characterized in that the micro chamber (11) comprises at least one trapping device (20) configured to capture at least one bacterial cell (12) in the micro chamber (11), and in that the sensor (18) is a field effect transistor-based ion sensor or a piezoresistive sensor.

2. The micro chamber device (1) of claim 1 , wherein the micro chamber (11), the fluid inlet (14) and the fluid outlet (16) are formed of a single material on the substrate (40).

3. The micro chamber device (1) of claim 1 or 2, wherein a lateral width (W) along the third axis (y) of the micro chamber (11) exceeds a plurality of bacterial cells, measured along a largest cell extension (c) of the largest bacterial cell size of the predetermined range of bacterial cell sizes, and wherein the at least one trapping device (20) is configured to accommodate a single bacterial cell (12), and wherein the at least trapping device (20) is arranged in the micro chamber (11) between the fluid inlet (14) and the fluid outlet (16).

4. The micro chamber device (1) of claim 3, wherein the fluid inlet (14) and the fluid outlet (16) are arranged on laterally opposite sides of the at least one trapping device (20).

5. The micro chamber device (1) of any of claims 3 or 4, wherein the trapping device (20) is a structure having a width (w) along the third axis (y) and a height (h) along the first axis (z) corresponding to the largest cell extension (c) of the predetermined range of bacterial cell sizes.

6. The micro chamber device (1) of any of claims 3-5, wherein the trapping device (20) has a concave surface (22) facing the fluid inlet (14), the concave surface (22) being configured to accommodate a single bacterial cell (12) and wherein the trapping device (20) further has a fluid channel (24) extending through the trapping device (20) in a direction from the concave surface (22) towards the fluid outlet (16) for directing a bacterial cell (12) towards the trapping device (20) to be accommodated by the concave surface (22).

7. The micro chamber device (1) of claim 1 or 2, wherein the micro chamber (11) further comprises a drain trench (26) for evacuating fluid from the micro chamber (11), the drain trench (26) extending along the first axis (z) from the micro chamber (11) through the substrate (40), and wherein the trapping device comprises nanowires (28) extending laterally across the micro chamber (11), such that the fluid inlet (14) and the fluid outlet (16) are on an opposite side of the nanowires (28) in relation to the drain trench (26), as seen along the first axis (z), the nanowires (28) further comprising the at least one trapping device (20) in the form of a nanowire net (20) having a lateral extension along the second axis (x) and the third axis (y) which lateral extension corresponds to a largest cell extension (c) of the predetermined range of bacterial cell sizes to accommodate a single bacterial cell (12) at the trapping device (20)..

8. The micro chamber device (1) of claim 7, wherein the trapping device (20) is surface- functionalized to cause loose attachment of the bacterial cell (12) to the trapping device (20).

9. The micro chamber device (1) of any of claims 1-8, wherein the fluid inlet (14) comprises a plurality of first openings having a first lateral width (D), configured to allow a predetermined range of bacterial cell sizes or smaller objects to pass through the first openings, and wherein the fluid outlet comprises a plurality of second openings having a second lateral width (d), configured to prevent bacterial cells (12) of the predetermined range of bacterial cell sizes or larger objects from passing through the second openings.

10. The micro chamber device (1) of claim 9, wherein the trapping device (20) comprises the first openings and the second openings.

11 . A test system (2) for performing antibiotic susceptibility tests on at least one bacterial cell (12), the test system (2) comprising at least one micro chamber device (1) according to any one of the claims 1-10, the test system (2) further comprising at least a first channel (34) fluidly connected to the fluid inlet (14) of the at least one micro chamber (11) for supplying a liquid to the micro chamber (11), at least a second channel (36) fluidly connected to the fluid outlet (16) of the at least one micro chamber (11) for evacuating the liquid from the micro chamber (11).

12. The test system (2) of claim 11 , wherein the test system (2) comprises a plurality of micro chamber devices (1), wherein at least some of the plurality of micro chamber devices (1) are differently dimensioned for bacterial cells (12) of different predetermined ranges of bacterial cell sizes.

13. The test system (2) of claim 11 or 12, further comprising a monitoring unit (38) for monitoring bacterial cell activity in the at least one micro chamber (11), the monitoring unit being electrically connected to the sensor (18) of the at least one micro chamber (11).

14. A method (3) for performing antibiotic susceptibility tests on bacterial cells (12) in the test system (2) according to any of claims 11-13 the method comprising: via the at least first channel (34), loading (S1) the at least one micro chamber (11) with a liquid bacterial sample comprising bacterial cells (12), capturing (S2), by the trapping device (20), at least one bacterial cell (12) in the at least one micro chamber (11), loading (S3) culturing media, with or without antibiotics, to each of the at least one micro chamber (11), and measuring (S4), by the sensor (18), at least one property of bacterial cell activity of the captured bacterial cell (12) in each of the at least one micro chamber (11).

15. The method (3) of claim 14, comprising closing (S5) the fluid inlet (14) and/or the fluid outlet (16) of the at least one micro chamber device (1) by closing the at least first channel (34) and/or second channel (36).

16. The method (3) of any of claims 14-15, wherein the property measured by the sensor (18) is metabolism-induced acidification of the culturing media and/or bacterial motion.