Devices, systems, and methods for performing antibiotic sensitivity testing

Abstract

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

Description

Technical Field

[0001] The present disclosure relates to a microchamber device for performing antibiotic susceptibility testing on bacterial cells. In particular, the present disclosure relates to a microchamber configured to capture at least one bacterial cell having a predetermined bacterial cell size range. The present disclosure also relates to a testing system including at least one such microchamber and a method for performing antibiotic susceptibility testing on bacterial cells in the testing system.

[0002] background

[0003] Rapid and precise treatment of infectious diseases is crucial to avoid costly and potentially fatal complications that lead to increased patient morbidity, drug side effects, etc.

[0004] Performing antibiotic susceptibility testing (AST) before introducing drug therapy can ensure that the correct antibiotic is selected for precise treatment of the patient. It can also prevent the development of antibiotic resistance due to the misuse of the latest versions of drugs.

[0005] Common approaches to AST use bacterial / cell growth or behavior as indicators of the presence of different antibiotics, such as the classic phenotypic antibiotic resistance analysis method performed by disk diffusion tests.

[0006] However, traditional methods that examine the growth of cells into colonies in culture in the presence of different drugs typically take more than two days, making them unable to guide precise medical treatment during the early, often critical stages of disease development.

[0007] Furthermore, obtaining a bacterial sample with sufficient bacterial count for testing is a major challenge for many infectious diseases, which often requires a pre-culture step of at least one day before starting AST.

[0008] Furthermore, these conventional methods are based on the detection of bacterial population behavior. They are limited to samples containing subpopulations of slow-growing or non-growing bacteria, which can resume growth after antibiotic removal and lead to relapse of infection. To be able to study the impact of these slow-growing or non-growing bacteria on infection treatment, new devices and methods for AST at the single-cell level are needed.

[0009] Overview

[0010] Therefore, it is an object of the present disclosure to provide an improved device for antibiotic susceptibility testing. More specifically, it is an object of the present disclosure to provide a microchamber, system and method for more accurately testing and shortening the testing time of antibiotics on single or multiple bacterial cells.

[0011] According to a first aspect of the present disclosure, the object is at least partially achieved by a microchamber device according to claim 1 .

[0012] Therefore, a microchamber device for performing antibiotic sensitivity testing on bacterial cells of a predetermined range of bacterial cell sizes is provided. The microchamber device is arranged on a substrate, the substrate extending laterally in a plane including a second axis and a third axis orthogonal to the second axis, the plane being orthogonal to the first axis. The microchamber device includes a fluid inlet and a fluid outlet in fluid communication with the microchamber, and an integrated sensor arranged in the microchamber, the sensor being configured to measure a property of bacterial cell activity in the microchamber. The microchamber includes at least one capture device, the at least one capture device being configured to capture at least one bacterial cell in the microchamber. The sensor is an ion sensor based on a field effect transistor or a piezoresistive sensor.

[0013] Microchambers can be manufactured at low cost in conventional semiconductor chip foundries using conventional semiconductor manufacturing techniques. Thus, the microchambers can be mounted on glass or semiconductor substrates or chips. The microchambers are configured for high-throughput bacterial collection microfluidics for multiplex rapid antibiotic susceptibility testing. The term "multiplex" should be understood herein to allow simultaneous testing of different antibiotics in different microchambers of a test system comprising a microchamber device. The small size of the semiconductor device facilitates direct testing of bacterial samples without the need for any pre-incubation.

[0014] For reference, different types of bacterial cells have different sizes, ranging from a few hundred nanometers to about 10 microns. Due to the low cost of manufacturing microchambers, the disclosed test system can be manufactured and stored by the laboratory in different sizes, intended for use with different ranges of bacterial cell diameters. Therefore, when a drug is to be tested on a predetermined bacterial cell type, the size of the bacterial cell type can be determined, and a test system of corresponding size with a microchamber sized for the relevant range of bacterial cell sizes can be selected from the stored test systems. In particular, the maximum and minimum cell extensions for the predetermined bacterial cell type are determined. The relevant range is understood in this context to mean a range of cell sizes that includes the determined cell size.

[0015] Alternatively, if the bacterial cell type is unknown, a test system can be selected that includes microchambers of different sizes, wherein the different microchamber sizes are targeted to different predetermined ranges of cell sizes. Thus, time is saved because the bacterial cells do not need to be studied prior to antibiotic susceptibility testing.

[0016] A predetermined range of cell sizes is understood herein to mean a range of cell sizes that allows for the separation / detection of a limited range of bacterial cells / sizes (i.e., cells of similar size but not necessarily identical size). Thus, a particular microchamber will be designed to accommodate cells of a limited size range for testing. Additional microchambers can simultaneously be designed and used for other predetermined ranges of bacterial cell sizes, thereby allowing blind testing of cells of unknown type and size, as some of the microchambers in the microchamber test system will correctly determine the size for the unknown cell type.

[0017] The at least one bacterial cell being "trapped" in the microchamber is understood to mean that the bacterial cell is contained by the trapping device or is loosely held by the trapping device or is prevented from leaving the microchamber by the trapping device. In this context, loosely held is understood to mean that the bacterial cell is able to move in its position and at the same time is prevented from leaving the trapping device.

[0018] The fluid inlet is arranged to introduce fluid into the microchamber. The fluid can be a fluid containing a bacterial sample (i.e., bacterial cells) or a fluid comprising a culture medium for nourishing bacterial cells. The fluid comprising the culture medium may or may not comprise an antibiotic for testing bacterial cells.

[0019] The fluid introduced into the microchamber can be discharged through the fluid outlet. Before the bacterial sample in the microchamber is tested, the fluid inlet and / or fluid outlet can be closed by sealing fluid such as oil, or by closing a valve with which the fluid inlet and fluid outlet can be in fluid communication.

[0020] The capture devices according to the present disclosure can be configured in different ways to capture a single bacterial cell or multiple bacterial cells in a microchamber. A capture device can be configured to capture only a single cell. Alternatively, a capture device can be configured to capture multiple cells in a microchamber. However, if multiple bacterial cells are to be tested, multiple single-cell capture devices can also be arranged in a microchamber. Thus, the behavior of single bacterial cells in some microchambers of a test system including a microchamber device of the present disclosure can be studied relative to multiple bacterial cells in other microchambers.

[0021] The sensor can be an integrated nanoscale semiconductor electronic sensor for real-time monitoring of bacterial activity. Therefore, the sensor may be an ion sensor based on a field effect transistor, such as a potentiometric ion sensor, which monitors the metabolism of bacterial cells by measuring the metabolically induced acidification of the culture medium. Alternatively, the sensor can be an integrated piezoresistive sensor that can monitor bacterial cell movement via the piezoresistive effect of materials such as silicon (Si). Introducing effective drugs (antibiotics) can inactivate bacterial cells. From the perspective of sensing, pH potential sensing using a field effect transistor is much more reliable than directly measuring the resistance (or resistivity) of the culture medium, because the contribution from the solid-liquid interface between the electrode / liquid may produce a large random error in the resistance measurement. In addition, directly monitoring the change of physiological parameters (such as pH or movement of the culture medium) may be much faster than optical monitoring of cell growth because it does not rely on visible morphological changes for signal generation. Using highly sensitive and reliable field effect transistor-based ion sensors or piezoresistive sensors, monitoring physiological parameters can potentially describe the drug response of bacteria in real time. The effect of the drug can be immediately detected from the reduced electrical signal generated by cell metabolism or cell movement. Antibiotic sensitivity testing is all-electrical, based on current-voltage measurement using conventional equipment. The sensor is "integrated" meaning that it could have been manufactured using conventional semiconductor processing techniques.

[0022] This article discloses the capture and drug testing of single bacterial cells. As described in the Background section above, some cell populations contain subpopulations of slow-growing or non-growing cells. When conventional antibiotic tests are performed on groups of cells, these cells are missed. The single-cell capture and testing described herein allows the study of single-cell behavior, and thus also the behavior of these slow-growing or non-growing cells.

[0023] Alternatively, the microchamber, fluid inlet, and fluid outlet are formed from a single material on a substrate. Any microstructure disclosed herein (such as the walls and roof of the microchamber and any connecting channels (e.g., inlet and outlet)) can be fabricated using conventional techniques using polydimethylsiloxane (PDMS) or glass on a semiconductor or glass substrate. This greatly simplifies fabrication.

[0024] Optionally, the lateral width of the microchamber along the third axis (y) exceeds the plurality of bacterial cells as seen along a maximum cell extension (c) of a maximum bacterial cell size in a predetermined range of bacterial cell sizes, and wherein the at least one capture device is configured to accommodate a single bacterial cell, and wherein the at least one capture device is arranged in the microchamber between the fluid inlet and the fluid outlet.

[0025] In one example, the lateral width of the microchamber allows bacterial cells to flow from the fluid inlet to the fluid outlet along with the fluid containing the bacterial cells. At least one capture device is disposed between the fluid inlet and the fluid outlet and is configured to receive / capture a single bacterial cell. Thus, the capture device is substantially sized to capture a bacterial cell. In other words, the capture device is large enough to capture a bacterial cell, but when a bacterial cell has been captured by the capture device, the capture device is full (i.e., occupied) and may no longer contain any bacterial cells.

[0026] Optionally, the fluid inlet and the fluid outlet are arranged on laterally opposite sides of the at least one capturing device.Thereby, the at least one capturing device is arranged in the fluid flow containing the bacterial sample.

[0027] Optionally, the capture device comprises a structure having a lateral width along the third axis and a height along the first axis corresponding to a maximum cell extension within a predetermined range of bacterial cell sizes. Thus, the lateral extension and height are configured to match the maximum extension of a bacterial cell. The capture device of this example can be a structure disposed on a substrate. The structure can be formed from the same single material as the microchamber, fluid inlet, and fluid outlet.

[0028] Optionally, the capture device has a concave surface facing the fluid inlet, the concave surface being arranged to accommodate a single bacterial cell, and wherein the capture device further has a fluid channel extending through the capture device in a direction from the concave surface towards the fluid outlet, for guiding bacterial cells towards the capture device to be accommodated by the concave surface.

[0029] Thus, the concave surface can function as a bowl in which individual bacterial cells can be collected and contained. A fluid channel extending through the capture device also provides a fluid flow through the capture device that directs bacterial cells from a liquid bacterial sample flowing into the microchamber toward the concave bowl-shaped surface of the capture device. The fluid channel can be configured to be small enough to prevent bacterial cells from entering the fluid channel, yet large enough to allow fluid to flow therethrough.

[0030] Optionally, the microchamber further comprises a drain groove for draining fluid from the microchamber, the drain groove extending from the microchamber through the substrate along the first axis, and wherein the capture device comprises a nanowire extending laterally across the microchamber such that the fluid inlet and the fluid outlet are on opposite sides of the nanowire relative to the drain groove, the nanowire further comprising the at least one capture device in the form of a nanowire mesh, the nanowire mesh having a lateral extension along the second axis and the third axis corresponding to a maximum cell extension of the predetermined range of bacterial cell sizes to accommodate a single bacterial cell at the capture device.

[0031] Thus, during the loading of the bacterial cells into the microchamber, a fluid containing the bacterial sample / cells is introduced into the microchamber via the fluid inlet and discharged via the fluid outlet. After a predetermined period of time, the microchamber is loaded. Thereafter, the fluid outlet can be closed. The microchamber can be provided on a substrate. A discharge groove can be etched through the substrate and can be fluidically connected to a pipe that can include an openable valve. After loading the microchamber and closing the fluid outlet, culture medium can be introduced into the microchamber via the fluid inlet and discharged via the discharge groove. When the culture medium is caused to flow through the microchamber, single bacterial cells will be captured / stopped on the capture device / nanowire mesh, while the remaining bacterial cells in the microchamber will be washed away via the discharge groove.

[0032] The nanowires and nanowire meshes are understood to be formed by conventional semiconductor processing techniques combined with the formation of drainage trenches through the substrate. Thus, the nanowires and nanowire meshes are monolithic components integrated with the substrate. The nanowires and nanowire meshes can have a cross-sectional diameter ranging between approximately five nanometers and several hundred nanometers.

[0033] Optionally, the capture device is surface functionalized to allow loose attachment of bacterial cells to the capture device. The capture device can be surface functionalized by conventional means, such as by a stable monolayer of the organic molecule ((3-aminopropyl)triethoxysilane (APTES). Functionalization renders the nanowire mesh biocompatible and improves electronic cell-sensor coupling. The loose attachment of bacterial cells allows the bacterial cells to move about their location but prevents the bacterial cells from leaving the capture device.

[0034] In this example, the capture device can include a sensor. The sensor can be a piezoelectric sensor. Thus, the movement of bacterial cells on the nanowire mesh causes vibrations in the nanowire mesh / capture device, which generates an electrical signal that can be measured to study bacterial movement.

[0035] Optionally, the fluid inlet comprises a plurality of first openings having a first lateral width configured to allow objects within a predetermined range of bacterial cell size or smaller to pass through the first openings, and wherein the fluid outlet comprises a plurality of second openings having a second lateral width configured to prevent objects within a predetermined range of bacterial cell size or larger from passing through the second openings.

[0036] The plurality of first openings enables filtering of large particles or cells and allows rapid flow of fluid into the microchamber via the fluid inlet. The plurality of second openings also enables cells with a predetermined cell size range to be retained in the microchamber and allows rapid flow of fluid out of the microchamber via the fluid outlet.

[0037] Optionally, the capture device includes a first opening and a second opening. The first opening prevents larger particles from entering the microchamber. The second opening can serve as a filter to prevent bacterial cells in the fluid containing the bacterial sample from leaving the microchamber. Thus, the first opening and the second opening serve as a capture device for capturing multiple bacterial cells in the microchamber.

[0038] According to a second aspect of the present disclosure, the object is at least partially achieved by a testing system according to claim 11 .

[0039] Therefore, a test system for performing an antibiotic sensitivity test on at least one bacterial cell is provided, the test system comprising at least one microchamber device according to any one embodiment of the first aspect of the present disclosure, the test system further comprising at least a first channel fluidically connected to a fluid inlet of the at least one microchamber for supplying liquid to the microchamber, and at least a second channel fluidically connected to a fluid outlet of the at least one microchamber for discharging liquid from the microchamber.

[0040] Optionally, the testing system comprises a plurality of microchamber devices, wherein at least some of the plurality of microchamber devices have different sizes for bacterial cells within different predetermined ranges of bacterial cell sizes.

[0041] Therefore, the test system can include microchambers of different sizes, where the sizes of the different microchambers are targeted at corresponding predetermined ranges of cell sizes. This allows for so-called blind testing of unknown bacterial cell types whose sizes have not yet been determined. Blind testing can save time because the bacterial cells do not have to be identified or measured to understand which antibiotics are effective against them.

[0042] The test system may include at least one microchamber array interconnected by a first and a second channel. Each microchamber array may be interconnected by a corresponding first channel and a second channel. During the test, each microchamber array may be supplied with different culture media, i.e., with or without antibiotics or with different antibiotics, to achieve multiple testing of antibiotics. The fluid inlet and fluid outlet of the microchamber included in the test system may be closed by closing the first channel and the second channel. The fluid inlet and the fluid outlet may be closed by closing the valve connected to the first channel and the second channel. Alternatively, the fluid inlet and the fluid outlet may be closed by filling the first channel and the second channel with a sealing fluid (e.g., oil). The sealing fluid may provide electrical insulation between the microchambers to improve the accuracy and reliability of the sensor measurements.

[0043] Optionally, the testing system further comprises a monitoring unit for monitoring the activity of bacterial cells in the at least one microchamber, wherein the monitoring unit is electrically connected to the sensor of the at least one microchamber.

[0044] The monitoring unit can be electrically connected to the test system to receive signals from each sensor of the microchamber included in the system. The monitoring unit can interpret the measurements of the sensors and provide a rapid representation of the response of the bacterial cells to the antibiotics supplied to the microchamber.

[0045] According to a third aspect of the present disclosure, the object is at least partially achieved by a method according to claim 13 .

[0046] Thus, a method for performing an antibiotic susceptibility test on at least one bacterial cell having at least one bacterial cell size within at least one predetermined range in a test system according to any embodiment of the second aspect of the present disclosure is provided. The method comprises loading the at least one microchamber with a liquid bacterial sample via the at least first channel, capturing at least one bacterial cell in the at least one microchamber by the capture device, loading culture medium with or without an antibiotic into each of the at least one microchamber, and measuring at least one property of bacterial cell activity of the cells captured in each of the at least one microchamber by the sensor.

[0047] Thus, the method allows for antibiotic susceptibility testing of bacterial cells of unknown size, such as an unknown bacterial cell type whose cell size has not yet been determined. This is achieved by using a test system comprising microchambers of different sizes, where the different microchambers are sized for different predetermined cell sizes. The correctly sized microchamber for the unknown cell type will provide relevant measurements from its respective sensor.

[0048] Thus, loading of the bacterial sample is performed by supplying a liquid bacterial sample through at least a first channel and into the microchamber via a fluid inlet. The liquid bacterial sample flows from the fluid inlet to the fluid outlet and into at least a second channel. In some examples, during a predetermined time period, it can be statistically guaranteed that at least one bacterial cell will be captured by at least one capture device of the microchamber. Thereafter, culture medium / liquid is loaded into the at least one microchamber for a predetermined time period, the culture medium may or may not contain an antibiotic to be tested on the at least one bacterial cell. In some examples, during the predetermined time period, it can be statistically guaranteed that at least one bacterial cell will be captured by at least one capture device of the microchamber. Unless prevented from leaving the microchamber by any second opening of the fluid outlet, loading of the culture medium will expel all or most of the bacterial cells not captured by the at least one capture device. The method may include stopping loading of the culture medium before measuring the cellular activity of the at least one bacterial cell. In this way, electrical measurement of the activity of the at least one bacterial cell by the sensor can be performed in a controlled liquid environment.

[0049] Optionally, the method comprises closing the fluid inlet and / or fluid outlet of the at least one microchamber device by closing the at least first channel and / or second channel. Thus, the control of the liquid environment in the at least one microchamber is further improved.

[0050] In one example, the method includes loading culture medium into each of at least one microchamber and draining the culture medium through the drainage groove, during which process bacterial cells will be captured / blocked by the nanowire mesh and the remaining bacterial cells in the microchamber 11 will be washed away via the drainage groove.

[0051] Optionally, the property measured by the sensor is metabolically induced medium acidification and / or bacterial motility, which properties are readily measurable by the sensors disclosed herein.

[0052] Optionally, the method may include draining the liquid bacterial sample via a drain groove. The fluid outlet may be sealed by a sealing fluid, and the liquid bacterial sample may be drained via a conduit that may be fluidically connected to the drain groove. Alternatively, the fluid outlet may be closed by closing a valve connected to the second channel.

[0053] As will be apparent to anyone skilled in the art, the above-mentioned aspects, the appended claims and / or the examples disclosed above and below may be combined with each other as appropriate.

[0054] Additional features and advantages are disclosed in the following description, claims, and drawings, and in part will be apparent to those skilled in the art or learned by practice of the disclosure as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Other objects, advantages, and features of the present disclosure will become apparent from the following description of one or more embodiments with reference to the accompanying drawings, in which:

[0057] Figure 1 A conceptual top view of a microchamber device according to the first aspect of the present disclosure is shown.

[0058] Figure 2 Shown are multiple single cell capture devices Figure 1 Example of a conceptual microchamber device.

[0059] Figure 3 Shown Figure 1 Cross-sectional side view of the microchamber device.

[0060] Figure 4 A top view of a microchamber device according to an example of the present disclosure is shown.

[0061] Figure 5 Shown Figure 4 Cross-sectional side view of the microchamber device.

[0062] Figure 6 A top view of a microchamber device according to an example of the present disclosure is shown.

[0063] Figure 7 Shown Figure 6 Cross-sectional side view of the microchamber device.

[0064] Figure 8 Shown Figure 6 Cross-sectional side view of the microchamber device.

[0065] Figure 9 A top view of a microchamber device according to an example of the present disclosure is shown.

[0066] Figure 10 Shown Figure 9 Cross-sectional side view of the microchamber device.

[0067] Figure 11 A top view of an exemplary testing system according to the second aspect of the present disclosure is shown.

[0068] Figure 12 Shown Figure 11 Cross-sectional side view of the test system.

[0069] Figure 13 A top view of an exemplary testing system according to the second aspect of the present disclosure is shown.

[0070] Figure 14 Shown Figure 13 Cross-sectional side view of the test system.

[0071] Figure 15 A top view of an exemplary testing system according to the second aspect of the present disclosure is shown.

[0072] Figure 16 A flow chart of a method according to the third aspect of the present disclosure is shown.

[0073] Figure 17 A flowchart of an example method according to the third aspect of the present disclosure is shown.

[0074] Detailed Description of Exemplary Embodiments of the Invention

[0075] The present disclosure is described in more detail below with reference to the accompanying drawings which illustrate examples of embodiments. The present disclosure should not be considered to be limited to the examples of embodiments described. Throughout the description, like numbers refer to like elements.

[0076] The terms used herein are for the purpose of describing specific aspects of the present disclosure only and are not intended to limit the present disclosure. As used herein, unless the context clearly indicates otherwise, the singular forms "a," "an," and "the" are intended to include the plural forms as well. Unless otherwise stated, 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 the present disclosure belongs.

[0077] Figure 1 A conceptual top view of a microchamber device 1 is shown, which is used to perform antibiotic sensitivity testing on bacterial cells 12 of a predetermined cell size according to the first aspect of the present disclosure. The microchamber device 1 is arranged on a substrate 40, which extends in a plane A, the plane A including a second axis x and a third axis y orthogonal to the second axis x, and the plane A is orthogonal to the first axis z. The microchamber device 1 includes a microchamber 11, a fluid inlet 14, a fluid outlet 16 in fluid communication with the microchamber 11, and a sensor 18 arranged in the microchamber 11. The sensor 18 is configured to measure a property of bacterial cell activity in the microchamber 11. The microchamber 11 also includes at least one capture device 20, which is configured to capture at least one bacterial cell 12 in the microchamber 11.

[0078] Any microstructures of the present disclosure, such as the walls and ceiling of the microchamber 11 and any connecting channels, can be fabricated on a semiconductor or glass substrate 40 by conventional techniques using a single material, such as polydimethylsiloxane (PDMS) or glass.

[0079] Figure 1 is a conceptual diagram in which the capture device 20 is illustrated as a box with a dotted outline. The capture device 20 according to the present disclosure can be configured in different ways to capture a single bacterial cell 12 or a plurality of bacterial cells 12 in a microchamber. One capture device 20 can be configured to capture only a single bacterial cell 12. Alternatively, the capture device 20 can capture a plurality of bacterial cells 12 in a microchamber 11. However, as Figure 2 As shown, for example, if multiple bacterial cells are to be tested, multiple single cell capture devices 20 can also be arranged in the microchamber 11, so that each capture device 20 captures one bacterial cell 12. The dashed box shown only indicates that there is a capture device 20 in the microchamber 11.

[0080] Figure 3 yes Figure 1 Cross-sectional side view of a mesochamber.

[0081] The arrows show the flow of liquid from the microchamber 11 via the fluid inlet 14, through the microchamber 11, and to the fluid outlet 16. The liquid can be, for example, a liquid bacterial sample containing bacterial cells 12, or a culture medium containing nutrients for the bacterial cells. The culture medium may also contain or not contain a drug or antibiotic to be tested on the at least one bacterial cell 12 captured in the microchamber 11. The fluid inlet 14 and the fluid outlet 16 can include a first opening and a second opening, respectively, such that the fluid inlet 14 and the fluid outlet 16 are bounded by the respective first opening and second opening.

[0082] Different types of bacterial cells 12 have different sizes, ranging from hundreds of nanometers to tens of micrometers. Since the microchambers 11 are inexpensive to manufacture, the disclosed test system 2 comprising a plurality of microchamber devices 1 can be manufactured and stored by a laboratory in different sizes for different bacterial cell diameters. Therefore, when a drug is to be tested on a predetermined bacterial cell type, the size of the bacterial cell type can be determined, and a test system 2 of the corresponding size (to be described below) can be selected from the stored test systems 2 for antibiotic sensitivity testing. In particular, the maximum cell extension and the minimum cell extension of the predetermined bacterial cell type are determined. Alternatively, if the bacterial cell type is unknown, a test system 2 comprising microchambers 11 of different sizes can be selected, wherein the sizes of the different microchambers 11 are for different predetermined cell sizes. Therefore, time is saved because the bacterial cells 12 do not need to be studied before antibiotic sensitivity testing.

[0083] The sensor 18 can be a nanoscale semiconductor electronic sensor 18 for monitoring bacterial cell activity in real time. The sensor 18 can be integrated on a substrate. Thus, the sensor 18 can be an ion sensor 18 based on a field effect transistor, which is configured to monitor bacterial cell metabolism by measuring the metabolically induced acidification of the culture medium. Alternatively, the sensor 18 can be a piezoresistive sensor 18, which can monitor bacterial cell movement via the piezoresistive effect of a material such as silicon (Si). The introduction of an effective drug (antibiotic) can inactivate the bacterial cells 12. The effect of the drug can be detected by a reduced electrical signal generated by bacterial cell metabolism / activity. Antibiotic sensitivity testing is all electrical and based on current-voltage measurements using conventional equipment.

[0084] An example of a capture device is shown in Figure 4 , which shows a top view of the microchamber device 1. The transverse width W of the microchamber 11 along the third axis y exceeds a plurality of bacterial cells measured along a maximum cell extension c of a maximum bacterial cell size in a predetermined range of bacterial cell sizes. At least one capture device 20 is configured to contain a single bacterial cell 12. The at least one capture device 20 is arranged in the microchamber 11 between the fluid inlet 14 and the fluid outlet 16.

[0085] The lateral width W of the microchamber 11 allows the bacterial cells 12 to flow from the fluid inlet 14 to the fluid outlet 16 together with the fluid (e.g., a bacterial sample) containing the bacterial cells 12. At least one capture device 20 or capture devices 20 is arranged between the fluid inlet 14 and the fluid outlet 16, and each of the at least one capture device 20 is configured to capture a single bacterial cell 12. Therefore, the capture device 20 substantially corresponds in size to the bacterial cell 12. In other words, the capture device 20 is large enough to accommodate the bacterial cell 12, but when one bacterial cell 12 has been captured by the capture device 20, the capture device 20 is full / occupied and cannot accommodate any more bacterial cells 12.

[0086] The fluid inlet 14 and the fluid outlet 16 are arranged on laterally opposite sides of the at least one capturing device 20. Thus, during loading of the bacterial sample into the microchamber 11, the at least one capturing device 20 is arranged in the fluid flow containing the bacterial sample.

[0087] The capture device 20 may be a structure having a transverse width w along the third axis y and a height h along the first axis z, such as Figure 5 As shown, it is configured to capture a single bacterial cell 12. Therefore, the lateral width w and height h are configured to have a maximum extension corresponding to the largest cell size in the predetermined cell size range.

[0088] The capture device 20 may also have a concave surface 22 facing the fluid inlet, the concave surface 22 being arranged to receive a single bacterial cell 12. The capture device 20 also has a fluid channel 24 extending through the capture device in a direction from the concave surface 22 toward the fluid outlet 16 for directing the bacterial cell 12 toward the capture device 20 to be received by the concave surface 22. Thus, the concave surface 22 may function as a bowl in which the single bacterial cell 12 may be collected. The fluid channel 24 also provides a fluid flow through the capture device 20 during use, which flow directs the bacterial cell 12 toward the capture device 20 to be received / captured by the concave bowl-shaped surface 22 of the capture device 20.

[0089] Figure 6Another example of a capture device 20 is shown, which shows a microchamber 11 including a drain channel 26 for draining fluid from the microchamber 11, the drain channel 26 extending from the microchamber 11 through a substrate 40 along a first axis z. The capture device 20 includes a nanowire 28 extending laterally across the microchamber 11, such that the fluid inlet 14 and the fluid outlet 16 are on opposite sides of the nanowire 28 relative to the drain channel 26, as seen along the first axis z. The nanowire 28 also includes at least one capture device 20 in the form of a nanowire mesh 20 having a lateral extension along the second axis x and the third axis y corresponding to a maximum cell size extension c of a predetermined range of bacterial cell sizes to accommodate a single bacterial cell 12 at the capture device 20.

[0090] Figure 6 Also shown are first and second channels 34, 36, which respectively deliver fluid to and collect fluid from the microchamber 11. The first and second channels 34, 36 are included in the test system 2 according to the second aspect of the present disclosure, which will be described in more detail below.

[0091] During the loading of the bacterial cells 12 into the microchamber 11, the fluid containing the bacterial sample / bacterial cells 12 is introduced into the microchamber 11 via the fluid inlet 14 and discharged via the fluid outlet 16. Figure 7 When the microchamber 11 is loaded, the fluid outlet 16 can be closed, as shown. Figure 8 . The fluid outlet 16 can be closed by closing a valve (not shown) connected to the second channel 36. The discharge groove 26 can be etched through the substrate 40 and can be fluidically connected to a pipe (not shown) on the opposite side of the substrate 40 relative to the microchamber 11. The dotted line 42 represents an interface with the pipe and indicates that the discharge groove 26 can be closed by a valve connected to the pipe (not shown) or by filling the pipe with a sealing fluid 32. After loading the microchamber 11 and closing the fluid outlet 16, the culture medium can be introduced into the microchamber 11 via the fluid inlet 14 and discharged via the discharge groove 26, as shown. Figure 8 As shown by the arrows in , during this process, the bacterial cells 12 will be captured / blocked by the nanowire mesh, and the remaining bacterial cells 12 in the microchamber 11 will be washed away via the drain groove 26 .

[0092] The nanowires 28 and nanowire mesh / trap device 20 are understood to be formed by conventional semiconductor processing techniques in conjunction with forming drain trenches 26 through the substrate 40. Thus, the nanowires 28 and nanowire mesh / trap device 20 are monolithic components that are integrated with the substrate 40. The nanowires 28 and trap device 20 may have a cross-sectional diameter ranging between approximately five nanometers and several hundred nanometers.

[0093] The exemplary capture device 20, exemplified by a nanowire mesh, can be surface functionalized to allow the bacterial cells 12 to loosely attach to the nanowire mesh. The nanowire mesh 20 can be surface functionalized by conventional means, such as by a stable monolayer of the organic molecule ((3-aminopropyl)triethoxysilane (APTES). Functionalization renders the nanowire mesh biocompatible and can improve electronic coupling between the bacterial cells 12 and the sensor 18.

[0094] In this example, the capture device 20 in the form of a nanowire mesh may include a sensor 18. The sensor 18 may be a piezoelectric sensor integrated into the nanowires 28. Thus, the movement of the captured bacterial cells 12 on the nanowire mesh / capture device 20 causes vibrations in the nanowire mesh / capture device 20, which generate electrical signals through the piezoelectric effect, which can be measured by the sensor 18 to study bacterial movement.

[0095] exist Figure 9 In another example shown, the fluid inlet 14 may include a plurality of first openings having a first lateral width D configured to allow bacterial cells 12 within a predetermined range of sizes or smaller to pass through the first openings. The fluid outlet 16 may also include a plurality of second openings having a second lateral width d configured to prevent bacterial cells 12 within a predetermined range of sizes or larger from passing through the second openings.

[0096] The plurality of first openings allow for filtering of large particles or cells and allow fluid to flow rapidly into the microchamber 11 via the fluid inlet 14. The plurality of second openings also enable bacterial cells 12 having a predetermined range of bacterial cell sizes or smaller to remain within the microchamber 11 and allow fluid to flow rapidly out of the microchamber 11 via the fluid outlet 16.

[0097] In this exemplary embodiment, the capture device 20 includes a first opening and a second opening. Thus, the capture device 20 corresponds to the entire microchamber device 1, including the first opening and the second opening. The second opening can act as a filter to prevent bacterial cells 12 in the fluid containing the bacterial sample from leaving the microchamber 11. Thus, the first opening and the second opening are used to capture a plurality of bacterial cells in the microchamber 11.

[0098] Figure 10 yes Figure 9 As described above, in order to close the microchamber 11, the fluid inlet 14 and the fluid outlet 16 can be closed by closing the shut-off valves (not shown) connected to the first channel 34 and the second channel 36. Alternatively, the fluid inlet 14 and the fluid inlet 16 can be closed by filling the first channel and the second channel with a sealing fluid 32 (e.g., oil), as shown in FIG. Figure 10As shown in the dashed area 32 in FIG. The microchambers 11 sealed by the sealing fluid 32 can produce more accurate measurements by the sensor 18 because the microchambers 11 can be electrically isolated from each other by the sealing fluid 32.

[0099] like Figure 6-Figure 8 As shown in FIG, the first opening and the second opening can advantageously be used as a filter in combination with the nanowire mesh capture device 20. Thus, during bacterial loading, a plurality of bacterial cells 12 will be captured in the microchamber 11. During subsequent culture medium loading, a single bacterial cell 12 will be captured by the nanowire mesh capture device 20, and the remaining bacterial cells 12 in the microchamber 11 will be washed away via the drain groove 26.

[0100] The second aspect of the present disclosure is as follows Figure 11-13 shown. Figure 11 A test system 2 for performing antibiotic susceptibility testing on at least one bacterial cell 12 having at least one predetermined range of bacterial cell sizes is shown. The test system 2 includes at least one microchamber device 1 according to an embodiment of the first aspect of the present disclosure. The test system 2 also includes at least a first channel 34 fluidically connected to the fluid inlet 14 of the at least one microchamber 11 for supplying liquid to the microchamber 11. The test system 2 also includes at least a second channel 36 fluidically connected to the fluid outlet 16 of the at least one microchamber 11 for draining liquid from the microchamber 11.

[0101] The testing system 2 may include a plurality of microchamber devices 1 , wherein at least some of the plurality of microchamber devices 1 have different sizes relative to each other for testing bacterial cells 12 of different predetermined bacterial cell size ranges.

[0102] Thus, the test system 2 can include microchambers 11 of different sizes, wherein the sizes of the different microchambers 11 are targeted at different predetermined ranges of bacterial cell sizes, which allows for so-called blind testing of unknown bacterial cell types whose sizes have not yet been determined. Blind testing can save time because it is not necessary to identify or measure the bacterial cells 12 to understand which antibiotics are effective against the cells.

[0103] The testing system 2 may include at least one array 44 of microchamber devices 1 interconnected by at least a first channel 34 and a second channel 36 . Figure 11Three microchambers 11 in an array 44 of microchamber devices 1 are shown. However, any number of microchamber devices 1 can be included in the array. An array is defined as a group of microchamber devices 1 interconnected by the same first channel 34 and the same second channel 34. The test system 2 can also include multiple arrays 44 of microchamber devices, each array 44 of microchamber devices 1 being interconnected by a corresponding first channel 34 and a second channel 36. During the test, each array 44 of the microchamber device 1 can be supplied with a different culture medium, i.e., with or without antibiotics, or with different antibiotics. The fluid inlet 14 and the fluid outlet 16 of the microchamber 11 included in the test system 2 can be closed by closing the first channel 34 and the second channel 36. The fluid inlet 14 and the fluid outlet 16 can be closed by closing the valves (not shown) connected to the first channel 34 and the second channel 36. Alternatively, the fluid inlet 14 and the fluid outlet 16 can be closed by filling the first channel 34 and the second channel 36 with a sealing fluid (e.g., oil).

[0104] It is worth noting that Figure 11 The microchamber device 1 shown depicts Figure 1-Figure 3 Therefore, the fluid inlet 14 and the fluid outlet 16 of the microchamber 11 may also include a first opening and a second opening, respectively. Figure 9 Thus, the dotted box representing the capture device may represent a structure protruding from the substrate 40, as shown. Figure 4 and Figure 5 As shown, or a nanowire network extending over the drain groove 26, as Figure 6-Figure 8 The structure and nanowire network are as described above.

[0105] Figure 12 Shown Figure 11 1 is an exemplary side view of a testing system 2 , wherein liquid is shown entering the microchamber 11 from a first channel 34 and exiting the microchamber 11 via a fluid outlet 16 to a second channel 36 .

[0106] Figure 13 and Figure 14 Shown include Figure 9 and Figure 10 . The capture device 20 is understood here to be the entire microchamber 11, including the first and second openings of the respective fluid inlet 14 and fluid outlet 16. In this example, the capture device is a multi-cell capture device, wherein many bacterial cells are trapped in the microchamber 11 due to the second opening of the fluid outlet 16, which is configured to be too narrow for the bacterial cells 12 to leave the microchamber 11. As a result, a group of bacterial cells 12 are tested and measured.

[0107] Figure 15An example of three arrays 44 of microchamber devices 1 is shown. During use, each array can be provided with a culture medium of a different composition. Thus, one array can be provided with an antibiotic, while another array can be provided with culture medium without antibiotics. Furthermore, different antibiotics can be introduced into different arrays. Each array can also include microchamber devices 1 of different sizes, allowing for testing bacteria of unknown size, a so-called blind test.

[0108] The test system 2 may be disposed on a substrate 40. The test system may be configured to be fluidly connected to a source of liquid bacterial sample (not shown) and / or a source of liquid culture medium (not shown).

[0109] The test system 2 may further include a monitoring unit 38 electrically connected to the sensors 18 of at least one microchamber device 1. The monitoring unit 38 may be electrically connected to the sensors 18 of the test system 2 to receive a signal from each sensor 18 of the microchambers 11 included in the test system 2. The monitoring unit 38 may interpret the measurement results of the sensors 18 and provide a rapid representation of the response of the bacterial cells 12 to the antibiotic supplied to the microchamber 1.

[0110] The third aspect of the present disclosure is as follows Figure 16 , a flow chart of a method 3 for performing antibiotic sensitivity testing on at least one bacterial cell 12 in a test system 2 according to any embodiment of the second aspect of the present disclosure is depicted. Unless otherwise specified, actions S2 and S3 are not performed in any particular order relative to each other. Method 3 comprises:

[0111] - loading S1 at least one microchamber 11 with a liquid bacterial sample containing bacterial cells 12 via at least a first channel 34,

[0112] - capturing S2 at least one bacterial cell 12 in at least one microchamber 11 by a capturing device 20,

[0113] - loading S3 culture medium with or without antibiotics into each of at least one microchamber 11, and

[0114] - measuring S4 at least one property of bacterial cell activity of the trapped bacterial cells 12 in each of the at least one microchamber 11 by means of a sensor 18 .

[0115] Thus, loading of the bacterial sample ( S1 ) is performed by supplying a liquid bacterial sample into the microchamber 11 via the fluid inlet 14 via at least the first channel 34. The liquid bacterial sample flows from the fluid inlet 14 to the fluid outlet 16 and into at least the second channel 36 for a predetermined period of time. In some examples, it is statistically guaranteed that at least one bacterial cell 12 will be captured by the at least one capture device 20 of the microchamber 11 during the predetermined period of time. Subsequently, the liquid bacterial sample is emptied by loading the at least one microchamber 11 with culture medium / liquid ( S3 ). In some instances, the bacterial cells 12 are captured by the capture device 20 during the loading of the culture medium ( S3 ). The culture medium may or may not contain an antibiotic for testing on the at least one bacterial cell 12. The method may include stopping ( S6 ) the loading of the culture medium. In this way, the electrical measurement of the activity of the at least one bacterial cell by the sensor 18 can be improved by performing the measurement in a more controlled liquid environment.

[0116] Method 3 may include closing S5 the fluid inlet 14 and / or the fluid outlet 16 of at least one microchamber device 1. The fluid inlet 14 and / or the fluid outlet 16 may be closed by closing a valve (not shown) connected to at least the first channel 34 and at least the second channel 36 or by filling at least the first channel 34 and / or the second channel 36 with a sealing medium 32. Thus, the control of the liquid environment in the at least one microchamber 11 is further improved.

[0117] In one example, if Figure 17 As shown, after the action of loading S1 the microchamber 11 with the liquid bacterial sample, the second channel 36 is closed S5 and the culture medium is loaded S3 into each of the at least one microchamber 11. The loading S3 of the culture medium results in the capture / arrest S2 of the bacterial cells 12 on the nanowire mesh / capture device 20, while the culture medium is discharged through the discharge groove 26, as shown. Figure 8 As shown by the arrow in . During the loading of the culture medium S3, since the flow of the culture medium is perpendicular to the lateral extension of the nanowire mesh / capture device 20, a single bacterial cell will be captured by the nanowire mesh capture device 20, while the remaining bacterial cells 12 in the microchamber 11 will be washed away through the drainage groove 26. As described above, exemplary method 3 can optionally include stopping S6 the loading of the culture medium before measuring S4 the cell activity of at least one bacterial cell. As described above, before starting to measure the cell activity S4, the first channel 34 and the second channel 36 can optionally be closed S5.

[0118] The property measured by the sensor 18 may be metabolically induced acidification of the culture medium and / or bacterial motility.

Claims

1. A microchamber device (1) for performing antibiotic susceptibility testing on bacterial cells (12) within a predetermined range of bacterial cell sizes, the microchamber device (1) being arranged on a substrate (40) extending transversely in a plane (A) including a second axis (x) and a third axis (y) orthogonal to the second axis (x), the plane (A) being orthogonal to the first axis (z), the microchamber device (1) comprising a microchamber (11), a fluid inlet (14) and a fluid outlet (16) in fluid communication with the microchamber (11), and an integrated sensor (18) arranged in the microchamber (11), the sensor (18) being configured to measure a property of bacterial cell activity in the microchamber (11), and It is characterized by: The microchamber (11) includes at least one trapping device (20) configured to capture at least one bacterial cell (12) in the microchamber (11), and the sensor (18) is a field effect transistor-based ion sensor or a piezoresistive sensor.

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

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

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

5. The microchamber device (1) according to any one of claims 3 or 4, wherein the capture 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 maximum cell extension (c) of the bacterial cell size in the predetermined range.

6. The microchamber device (1) according to any one of claims 3 to 5, wherein: The capture 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 capture device (20) further has a fluid channel (24) extending through the capture device (20) in a direction from the concave surface (22) toward the fluid outlet (16) for guiding the bacterial cell (12) toward the capture device (20) to be accommodated by the concave surface (22).

7. The microchamber device (1) according to claim 1 or 2, wherein the microchamber (11) further comprises a drain groove (26) for draining fluid from the microchamber (11), the drain groove (26) extending from the microchamber (11) through the substrate (40) along the first axis (z), and wherein the capture device comprises a nanowire (28) extending laterally across the microchamber (11) such that the fluid inlet (14) and the fluid outlet (16) are on opposite sides of the nanowire (28) relative to the drain groove (26) as seen along the first axis (z), the nanowire (28) further comprising the at least one capture device (20) in the form of a nanowire mesh (20), the nanowire mesh having a lateral extension along the second axis (x) and the third axis (y), the lateral extension corresponding to the maximum cell extension (c) of the predetermined range of bacterial cell sizes to accommodate a single bacterial cell (12) at the capture device (20).

8. The microchamber device (1) according to claim 7, wherein the capture device (20) is surface functionalized to allow the bacterial cells (12) to loosely attach to the capture device (20).

9. The microchamber device (1) according to any one of claims 1 to 8, wherein: The fluid inlet (14) includes a plurality of first openings having a first lateral width (D), the first lateral width being configured to allow objects of a predetermined range of bacterial cell sizes or smaller to pass through the first openings, and wherein the fluid outlet includes a plurality of second openings having a second lateral width (d), the second lateral width being 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 microchamber device (1) according to claim 9, wherein the trapping device (20) comprises the first opening and the second opening.

11. A test system (2) for performing an antibiotic sensitivity test on at least one bacterial cell (12), the test system (2) comprising at least one microchamber device (1) according to any one of claims 1 to 10, the test system (2) further comprising at least a first channel (34) fluidically connected to a fluid inlet (14) of the at least one microchamber (11) for supplying liquid to the microchamber (11), and at least a second channel (36) fluidically connected to a fluid outlet (16) of the at least one microchamber (11) for discharging liquid from the microchamber (11).

12. The test system (2) according to claim 11, wherein the test system (2) comprises a plurality of microchamber devices (1), wherein at least some of the plurality of microchamber devices (1) have different sizes for bacterial cells (12) of different predetermined ranges of bacterial cell sizes.

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

14. A method (3) for performing antibiotic susceptibility testing on bacterial cells (12) in a test system (2) according to any one of claims 11 to 13, the method comprising: loading (S1) the at least one microchamber (11) with a liquid bacterial sample containing bacterial cells (12) via the at least first channel (34), capturing (S2) at least one bacterial cell (12) in the at least one microchamber (11) by the capturing device (20), loading (S3) a culture medium with or without antibiotics into each of the at least one microchamber (11), and At least one property of bacterial cell activity of the captured bacterial cells (12) in each of the at least one microchamber (11) is measured (S4) by the sensor (18).

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

16. The method (3) according to any one of claims 14-15, wherein the property measured by the sensor (18) is metabolically induced acidification of the culture medium and / or bacterial motility.

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