Monitoring device

Abstract

A monitoring device (1) comprises a microwell plate (10) comprising microwells (20) dimensioned to hold a respective biological entity and a channel plate (30) comprising at least one fluid channel (40, 42) in fluid connection with a fluid input port (11; 11A, 11B) and a fluid output port (13; 13A, 13B). An optode composition (25) is present in the microwells (20) and comprising an analyte-sensitive optode compound generating a detectable optical signal in response to an analyte. The channel plate (30) and the microwell plate (10) are slidably arranged together and movable relative to each other between a loading position, in which the at least one fluid channel (40, 42) is in fluid connection with at least a subset (21, 23) of the plurality of microwells (25), and a monitoring position, in which the channel plate (30) closes the at least a subset (21, 23) of the plurality of microwells (20).

Description

[0001] MONITORING DEVICE

[0002] TECHNICAL FIELD

[0003] The present invention generally relates to a monitoring device, and in particular to such a device capable of monitoring the response of individual biological entities to tunable chemical conditions.

[0004] BACKGROUND

[0005] Exposure to chemicals can introduce cellular toxicity, a phenomenon investigated extensively in toxicology through various metabolic proxies, such as oxygen (O2) respiration, changes in gene expression, or alterations in growth across biological entities. Traditional toxicological testing often involves segregating populations of organisms into “controls” and “exposed” groups, and statistically comparing their responses to incremental chemical concentrations to obtain dose-response relationships. This presumes uniform behavior across populations and potentially disregards the variability in responses among individuals. While conventional approaches, thus, offer cost-efficiency and broad acceptance, they inherently obscure heterogeneous responses to chemical perturbations. The implication of this is particularly acute in medical microbiology, where, for instance, subsets of bacterial cells known as “persisters” exhibit distinct resilience to antibiotic regimes. Another example is individual cancer stem cells driving the relapse of disease progression within predominantly healthy cellular environments. Similarly, in microbial ecology, chemically resistant cells may aid populations in tolerating and adapting to chemical perturbations, and microalgae can exhibit varying susceptibilities to infochemicals, such as pheromones and hormones, released by bacteria, with significant implications for their interaction with viruses and their role in ecosystems.

[0006] Du et al., Lap Chip, 2009, 9: 2286-2292 discloses a microfluidic device designed to perform multiplexed microfluidic reactions without pumps or values. The device contains a bottom plate with wells preloaded with many reagents. The wells are covered by a top plate acting as a lid. A fluidic path, composed of ducts in the bottom plate and wells in the top and bottom plates, is connected only when the top and bottom plates are aligned in a specific configuration.

[0007] Kannan et al., Biomicrofluidics, 2023, 17: 054105 discloses integration of a microfluidic device and widefield frequency domain fluorescence imaging lifetime microscopy (FD-FLIM) for single-cell oxygen consumption rate (OCR) measurements.

[0008] WO 2010111265 A1 discloses a device having a first surface having a plurality of first areas and a second surface having a plurality of second areas. The first surface and the second surface are opposed to one another and can move relative to each other from at least a first position where none of the plurality of first areas, having a first substance, are exposed to plurality of second areas, having a second substance, to a second position. When in the second position, the plurality of first and second areas, and therefore the first and second substances, are exposed to one another. The device further includes a series of ducts in communication with a plurality of first second areas to allow for a substance to be disposed in, or upon, the plurality of second areas when in the first position.

[0009] There is still a need to monitor the response of individual biological entities to different chemical conditions, particularly when using the individual biological entities as their own control.

[0010] SUMMARY

[0011] It is a general objective to provide a device capable of monitoring the response of individual biological entities to different chemical conditions, and in particular when using individual biological entities as their own control.

[0012] This and other objectives are met by embodiments of the present invention.

[0013] The present invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0014] An aspect of the invention relates to a monitoring device comprising a microwell plate comprising a plurality of microwells dimensioned to hold a respective biological entity and arranged in a first main surface of the microwell plate. The monitoring device also comprises a channel plate comprising at least one fluid channel arranged in a first main surface of the channel plate and in fluid connection with a fluid input port and a fluid output port in a loading position. The monitoring device further comprises an optode composition present in the microwells and comprising an analyte-sensitive optode compound generating a detectable optical signal in response to an analyte. The channel plate and the microwell plate are slidably arranged together and movable relative to each other between the loading position, in which the at least one fluid channel is in fluid connection with at least a subset of the plurality of microwells, and a monitoring position, in which the channel plate closes the at least a subset of the plurality of microwells. At least the portion of the microwell plate comprising the plurality of microwells is optically transparent and / or at least the portion of the channel plate aligned with the at least a subset of the plurality of microwells in the monitoring position is optically transparent. Another aspect of the invention relates to a method of monitoring biological entities. The method comprises loading biological entities into microwells of the monitoring device according to above and moving the channel plate relative to the microwell plate, or vice versa, to the monitoring position. The method also comprises directing input light into the at least a subset of the plurality of microwells, detecting, for each microwell of the at least a subset of the plurality of microwells comprising at least one biological entity, output light from the microwell, and generating, for each microwell of the at least a subset of the plurality of microwells comprising at least one biological entity, a reference value based on the detected output light. The method further comprises moving the channel plate relative to the microwell plate, or vice versa, to the loading position, flowing a fluid through the at least one fluid channel in the loading position, and moving the channel plate relative to the microwell plate, or vice versa, to the monitoring position. The method additionally comprises directing input light into the at least a subset of the plurality of microwells, detecting, for each microwell of the at least a subset of the plurality of microwells comprising at least one biological entity, output light from the microwell, and generating, for each microwell of the at least a subset of the plurality of microwells comprising at least one biological entity, a measurement value based on the detected output light. The method also comprises determining an analyte response of biological entities to the fluid based on the measurement value and the reference value determined for each microwell of the at least a subset of the plurality of microwells comprising at least one biological entity.

[0015] A further aspect of the invention relates to a method for producing a monitoring device. The method comprises generating, in a first substrate, a plurality of microwells dimensioned to hold a respective biological entity to form a microwell plate comprising the plurality of microwells in a first main surface of the microwell plate. The method also comprises generating, in a second substrate, at least one fluid channel to form a channel plate comprising the at least one fluid channel in a first main surface of the channel plate. The method further comprises depositing an optode composition in the plurality of microwells. The optode composition comprises an analyte-sensitive optode compound generating a detectable optical signal in response to an analyte. The method additionally comprises slidably arranging the channel plate and the microwell plate together with the first main surface of the channel plate facing the first main surface of the microwell plate so that the channel plate and the microwell plate are movable relative to each other between a loading position, in which the at least one fluid channel is in fluid connection with at least a subset of the plurality of microwells, and a monitoring position, in which the channel plate closes the at least a subset of the plurality of microwells. At least the portion of the microwell plate comprising the plurality of microwells is optically transparent and / or at least the portion of the channel plate aligned with the at least a subset of the plurality of microwells in the monitoring position is optically transparent.

[0016] The monitoring device of the invention can be used to investigate the response of biological entities to various chemical or abiotic conditions, such as for toxicological testing. The monitoring device enables each monitored biological entity to be used as its own control or reference. As a consequence, the response of a given biological entity to a chemical or abiotic condition can be determined relative to a reference or control condition, in which the same biological entity was not exposed to the chemical or abiotic condition. This means that the monitoring device can be used for such toxicological testing and monitoring for heterogenous populations of biological entities, i.e., also in cases where is a variability in responses among individual biological entities to a given chemical or abiotic condition.

[0017] BRIEF DESCRIPTION ON THE DRAWINGS

[0018] The embodiments, together with further objects and advantages thereof, may best be understood by referring to the following description taken together with the accompanying drawings, in which:

[0019] Fig. 1. SlipO2Chip™, a glass microfluidic device for repeated single-cell O2 respiration measurements under tuneable chemical microenvironments. (1A) Image of the fully assembled SlipO2Chip™. SlipO2Chip™ consists of two glass plates - a channel plate on the top and a microwell plate at the bottom, affixed together with neodymium magnets and secured within a 3D printed holder to facilitate controlled slipping of the top channel plate over the bottom microwell plate. An explosion diagram of the assembly of the frame and SlipC^Chip™ components is available in Fig. 2. (1 B) Close-up of the bottom microwell array within SlipO2Chip™ and the fluid access resulting from assembling the top channel plate and the microwell plate. Each channel has dedicated inlet and outlet ports accessible through the bottom microwell plate. (1 C) Fluorescein was introduced into the fully assembled SlipO2Chip™ to visualize the controlled slipping motion used to close microwells and test the efficacy of the liquid seal during manipulations. Note the retained fluorescein solution in the individual microwells upon closure. The fluorescein intensity across the microwell array is provided and sharp peak edges and zero intensity in areas outside the closed microwells indicate the liquid-tightness of the sealed microwells. (1 D) A schematic depiction of the microscopy imaging setup used to conduct ratiometric O2 measurements. UV light (peak excitation band 395 nm and half-band width of 25 nm) was provided via a SpectraX light source, which excited the 02-sensitive optode material Pt(ll) meso-tetra(pentafluorophenyl)porphine (PtTFPP) as well as a co-embedded reference dye. The resulting luminescence emission of PtTFPP and reference dye was captured by an RGB camera. During image-processing, the RGB image was split into red (-650 nm) and green (-512 nm) channels in order to obtain ratiometric measurements of O2 concentrations. (1 E) Fluorescence image of deposited PtTFPP within microwells. Red rings signified the deposition of PtTFPP and reference dye primarily at the edges of the microwell. Here shown is the red luminescence intensity (in gray scale) of the optode material at 0 % and 100 % air saturation, respectively. The area where the optode material was coated and the region of interest (ROI), which was segmented for subsequent O2 concentration measurements are both labeled. (1 F) Calibration of red / green ratiometric images across seven O2 concentrations and its description via the Stern-Volmer relationship (see Equation 1). Shown are the Stern-Volmer constant (ksv), and the goodness of linear fit (R2), used to derive O2 concentrations from RGB images.

[0020] Fig. 2. Explosion diagram of the 3D printed holder of SlipO2Chip™. This holder affixed the top channel plate and the bottom microwell plate of SlipC>2Chip™ and enabled for the accurate slipping motion of the top channel plate via a pair of micrometer screws and the frame part, which drove the top channel plate. Both glass plates were maintained in close proximity to each other through the careful placement of neodynium magnets. Four magnets were glued onto four corners of the top channel plate and perfectly fitted in the duct of the frame part. The other four magnets were placed underneath the bottom microwell plate and were movable along slipping manipulations.

[0021] Fig. 3. Verification of the gas-tightness of SlipO2Chip™. (3A) Cross-sectional visualization of the experiment conducted to ensure gas tightness of SlipO2Chip™. The microwell plate saturated with L1 media was placed into a customized gas control chamber. Half of the microwell plate was covered by PDMS film and the rest part was covered by a PDMS-PET film with the PET side facing the microwells. The microwell plate was first exposed to compressed air (CA, 100% air saturation) and, after a 10-min interval, 100% N2 gas was introduced into the chamber. During this stepwise exposure to different O2 concentrations, the O2 concentration within microwells was monitored every 2 min using automated microscopy and an RGB camera. (3B) Change of O2 concentrations in microwells during the 60-min gastightness experiment. Microwells sealed with a PET film successfully maintained 100% air saturation even after a 50-min exposure to N2. In contrast, microwells covered solely with a PDMS layer displayed a rapid decline in O2 after the surrounding was saturated with N2, reaching 0% O2 concentration. This difference underscores the role of the PET layer in ensuring the gas-tightness of the microwells.

[0022] Fig. 4. Schematic protocol of dry and wet etching protocols used in the production of SlipO2Chip™. (4A) Dry etching protocol used to generate the microwell plate. Reactive ion etching (RIE) of borosilicate glass was carried out using SF6 and SF6 / Ar plasma. Sputtered Al on the glass structure using patterned AZ- 10XT photoresist mask was used as a hard-mask for plasma etching. (4B) Wet etching protocol used to generate the channel structure. The wet etching of fused silica glass was carried out using 50% HF acid. Sputtered Mo on the glass structure using patterned S1813 photoresist mask was used as a hard-mask for the wet etching.

[0023] Fig. 5. Protocol to decorate microwells with O2 sensitive optode materials. A cocktail of PtTFPP, reference dye (MY), polystyrene and silicone rubber were dissolved in toluene and this optode chemistry was evenly spread on the microwell plate surface via a surface applicator tool. After solvent evaporation, a thin layer of optode composition was formed across the entire microwell surface and subsequently removed using a scalpel. This resulted in a thin layer deposition of optode composition at the sides and the bottom of the microwell.

[0024] Fig. 6. Experimental setup for calibrating deposited optode composition within microwells. Here shown is a custom gas chamber composed of the gas-impermeable material polyoxymethylene (POM). The excitation light used to stimulate optode composition was applied through the objective of an inverted microscope connected to an RGB camera for imaging. By connecting the chamber to a digital gas mixer consisting of 2 gas flow meters (High-precision thermal mass flow meter, Vbgtlin Instruments GmbH, CH), the flow rates of nitrogen gas (N2) and compressed air (CA) were tuned to saturate the chamber with different O2 concentrations. A multi-point O2 calibration was realized for each microwell plate. The actual O2 concentrations in the chamber were confirmed by inserting a commercial O2 sensor (FireSting, Pyroscience GmbH, DE).

[0025] Fig. 7. Schematic illustration of the coordinated opening and closing of SlipO2Chip™ for conducting single-cell O2 respiration measurements before and after chemical exposure. (7A) Cross section of SlipO2Chip™ with the top channel plate and the bottom microwell plate. In Step 1 , to load cells, a dense suspension of cells is introduced into the main channel, where gravity leads to their deposition into microwells. Cells located outside of microwells are removed by connecting the inlet to a low-pressure pump and flushing them out with media. In Step 2, cells are isolated within closed microwells by horizontally sliding the top channel plate, facilitating the measurement of single-cell O2 respiration through deposited optode materials. In Step 3, the top channel plate is slipped in the opposite direction to open microwells, facilitating the subsequent introduction of liquids such as fully oxygenated media or chemical solutions. The selection of these liquids is managed by a Y-shaped valve positioned outside of SlipO2Chip™. In Step 4, closing microwells for a second time allows for a repeated respiration measurement of the same cell, with the option of conducting this measurement under exposure to previously introduced chemicals, see Step 3. (7B) Exemplary data on O2 respiration of a single diatom cell before and after exposure to the chemical 2-heptyl-4-quinolone (HHQ) as outlined by steps 1-4. O2 respiration measurements were taken every 2 min over a span of 16 min. Rates of single-cell O2 respiration before and after introduction of HHQ were determined through linear fits of O2 consumption curves.

[0026] Fig. 8. Opening and closing SlipO2Chip™ twice has negligible effects on microalgae respiration. (8A) Time resolved O2 concentrations measured in 18 microwells containing a single-cell of the microalgae D. brightwellii. Curves depict dark O2 consumption for individual cells upon repeated opening and closing of microwells. Repeated respiration measurements are labeled as Respir. 1 , Respir. 2 and Respir. 3, respectively. Following each respiration measurements, cells were supplied with fully oxygenated media. (8B) Box and whisker plot of the pairwise comparison of single-cell O2 respiration rates obtained from three sequential opening and closing cycles. Boxes depict the mean and whiskers quartiles, and bars showing 1 % outliers of the O2 respiration rate data. Respiration rates were calculated from data presented in (8A) and statistically analyzed in a pairwise fashion using a Mann-Whitney U test. The resulting p- values between groups are indicated above the box plot and average percent changes compared to neighboring groups are indicated below box plots. (8C) Microscopy images of a representative microwell featuring a solitary cell with embedded optodes and reference dye (highlighted by a dashed ring). The images depict the red luminescence intensity at the beginning and end of each opening and closing cycle, providing a visual representation of the changes in O2 concentration resulting from single-cell respiration within the microwells. The natural autofluorescence of the immobilized cell is visible. This signal was not included in image processing as it lies outside the region of interest (ROI) used.

[0027] Fig. 9. Increasing concentrations of HHQ reduce the O2 respiration of single microalgae cells. (9A) Assessment of the single-cell O2 respiration of the microalgae D. brightwellii before and after exposure to HHQ (0, 5, 10, 20, 100 pM) as measured by SlipO2Chip™. In each HHQ exposure group, 26 to 71 single cells were measured (exact numbers displayed below). Boxes depict the mean and whiskers quartiles, and bars showing 1 % outliers of the O2 respiration rate data. Adjacent to each box, the normal distribution of the data is shown. (9B) The O2 respiration rates of 10 single-cells before and after HHQ exposure. These cells were randomly selected from the 5 pM exposure group. (9C) Dose-response relationship between the reduction of respiration rates (E) versus HHQ concentration. The reduction in respiration among investigated single cells was quantified by comparing respiration rates before and after exposure to HHQ. Here shown is this change for both single cells (individual black dots) and bulk measurements (non-filled dots). For single cells, the mean is shown as solid black dots and standard deviation is represented by error bars. Bulk measurements were only conducted once, thus no standard deviation is provided.

[0028] Fig. 10. Top view and cross-sectional view of a microwell plate according to an embodiment together with a close-up of a microwell according to an embodiment.

[0029] Fig. 11 . Bottom view and cross-sectional view of a channel plate according to an embodiment.

[0030] Fig. 12. The monitoring device in a cross-sectional view in the loading position (12A), the monitoring position (12B) and the open position (12C) according to an embodiment.

[0031] Fig. 13. The monitoring device in a view from above in the loading position.

[0032] Fig. 14. Top view and cross-sectional view of a microwell plate according to another embodiment.

[0033] Fig. 15. Bottom view and cross-sectional view of a channel plate according to another embodiment.

[0034] Fig. 16. The monitoring device in the loading position (16A) and the monitoring position (16B) according to another embodiment.

[0035] DETAILED DESCRIPTION

[0036] The present invention generally relates to a monitoring device, and in particular to such a device capable of monitoring the response of individual biological entities to tunable chemical conditions.

[0037] The monitoring device of the invention can be used to investigate the response of biological entities to various chemical or abiotic conditions, such as for toxicological testing. The monitoring device enables each monitored biological entity to be used as its own control or reference. As a consequence, the response of a given biological entity to a chemical or abiotic condition can be determined relative to a reference or control condition, in which the same biological entity is not exposed to the chemical or abiotic condition. This means that the monitoring device can be used for such toxicological testing and monitoring for heterogenous populations of biological entities, i.e., also in cases where is a variability in responses among individual biological entities to a given chemical or abiotic condition. As an illustrative example, the responses of cancer cells to a chemotherapeutic agent could be monitored by the monitoring device even if the cancer cell population is a mixture of persistent cancer cells resistant to the chemotherapeutic agent and cancer cells that are susceptible to the chemotherapeutic agent. As a consequence, such persistent cancer cells could be detected using the monitoring device even if they constitute a minute subpopulation of the cancer cells, which would not be identified when using separate “controls” and “exposed” cancer cell groups.

[0038] This monitoring device can be used for conducting measurements in the coordinated presence and absence of, for instance, chemical solutes. The monitoring device comprises a microwell plate and a channel plate movable relative to each other. In addition, the monitoring device features an array of individual microwells coated with an analyte-sensitive optode compound.

[0039] The monitoring device can be used, for instance, to measure respiration rates, a vital indicator of metabolism, both before and after chemical exposure within the same cell. Such a monitoring device will find broad applications in fields dealing with scarce sample volumes. This includes areas like clinical settings and medical diagnostics, where precise and sensitive measurements on a few, rare biological entities are indispensable. Furthermore, the monitoring device provides (eco)toxicologists and microbiologists with a refined tool to explore how cell heterogeneity influences the overall sensitivity of populations to chemicals and how this relates to the emergence of tolerant subpopulations. The monitoring device comprises a valuable tool for answering biological questions related to ecology, (eco)toxicology and microbiology at the single-biological entitylevel.

[0040] Further, the ability of the monitoring device to sequentially assess the effects of chemical substances on biological entities is advantageous when sample volumes are limited, such as clinical biopsies, studies involving rare microbial isolates, and toxicological studies wanting to address exposure effects while accounting for cell-to-cell variability.

[0041] With reference to Figs. 10-16, an aspect of the invention relates to a monitoring device 1 . The monitoring device 1 comprises a microwell plate 10, also referred to as multi-well plate, comprising a plurality of microwells 20 dimensioned to hold a respective biological entity and arranged in a first main surface 12 of the microwell plate 10. The monitoring device 1 also comprises a channel plate 30 comprising at least one fluid channel 40, 42 in a first main surface 34 of the channel plate 30 and in fluid connection with a fluid input port 11 ; 11 A, 11 B and a fluid output port 13; 13A, 13B in a loading position. An optode composition 25 is present in the microwells 20 and comprises an analyte-sensitive optode compound generating a detectable optical signal in response to an analyte. The channel plate 30 and the microwell plate 10 are slidably arranged together and movable relative to each other between the loading position, see Figs. 12A, 16A, in which the at least one fluid channel 40, 42 is in fluid connection with at least a subset 21 , 23 of the plurality of microwells 20, and a monitoring position, see Figs. 12B, 16B, in which the channel plate 30 closes the at least a subset 21 , 23 of the plurality of microwells 20. At least the portion of the microwell plate 10 comprising the plurality of microwells 20 is optically transparent and / or at least the portion of the channel plate 30 aligned with the at least a subset 21 , 23 of the plurality of microwells 20 in the monitoring position is optically transparent.

[0042] The monitoring device 1 , thus, comprises two plates 10, 30, the microwell plate 10 acting as a bottom plate with a plurality of microwells 20 therein and the channel plate 30 acting as a top plate or lid for the microwell plate 10. The microwell plate 10 and the channel plate 30 are slidably arranged together to movable relative to each other.

[0043] In an embodiment, the microwell plate 10 is stationary and the channel plate 30 can be slid on and relative to the microwell plate 10 between the loading position (Figs. 12A, 16A) and the monitoring position (Figs. 12B, 16B).

[0044] In another embodiment, the channel plate 30 is stationary and the microwell plate 10 can be slid below and relative to the channel plate 30 between the loading position (Figs. 12A, 16A) and the monitoring position (Figs. 12B, 16B).

[0045] In a further embodiment, both the microwell plate 10 and the channel plate 30 can be movable relative to each other so that they pushed or moved in opposite directions to move from the loading position (Figs. 12A, 16A) to the monitoring position (Figs. 12B, 16B), or vice versa.

[0046] Moving the channel plate 30 relative to the microwell plate 10, or vice versa, as referred to herein involves moving the channel plate 30 relative to the stationary microwell plate 10, or moving the microwell plate 10 relative to the stationary channel plate 30, or moving both the channel plate 30 and the microwell plate 10 relative to each other.

[0047] The relative movement between the microwell plate 10 and the channel plate 30 implies that the channel plate 30 will act as a lid for and thereby close the at least a subset 21 , 23 of the plurality of microwells 20 in the microwell plate 10 in the monitoring position. However, in the loading position, the at least one fluid channel 40, 42 is in fluid connection, i.e., aligned with, the least a subset 21 , 23 of the plurality of microwells 20. At this loading position, biological entities present in the microwells 20 can be exposed to, for instance, a chemical agent or abiotic condition, by flowing a fluid comprising the chemical agent through the at least one fluid channel 40, 42.

[0048] The monitoring position is then used to monitor the response of biological entities to control or reference conditions and to, for instance, the chemical agent or abiotic condition, which will be further described herein.

[0049] Abiotic condition as used herein include temperature, acidity, salinity that can be changed by adjusting the temperature, pH or ion concentration of the fluid perfused through the at least one fluid channel 40, 42.

[0050] The microwells 20 in the microwell plate 10 are dimensions to hold a respective biological entity or, less preferred, multiple, i.e., at least two biological entities. The microwells 20 also comprise an optode composition 25 comprising an analyte-sensitive optode compound, also referred to as analyte-specific optode compound herein. The analyte-sensitive optode compound generates a detectable optical signal in response to a particular analyte. In such a case, the biological entities present in the microwells 20 may produce and preferably release the analyte in response to the chemical agent or abiotic condition. The optical signal generated by the analyte-sensitive optode compound in the optode composition 25 thereby represents the response of the biological entities in the microwells 20 to control or reference conditions and to experimental conditions following exposure to the chemical agent or abiotic condition. As an example, the analyte-specific optode compound could be an oxygen (O2) sensitive optode compound employed to monitor the metabolic activity of individual biological entities in the microwells 20. The metabolic activities of individual biological entities in the microwells 20 could then be determined during control or reference conditions in the monitoring position prior to exposing the biological entities to any fluid including a chemical agent. In such an approach, the optical signals of individual biological entities are measured in the monitoring position to thereby get a respective representation of the metabolic activity of the individual biological entities during the control or reference condition. The monitoring device 1 is then put into the loading position to flow a fluid including a chemical agent to be tested on the biological entity through the at least one fluid channel 40, 42. The monitoring device 1 is thereafter put back into the monitoring position to anew measure optical signals of the individual biological entities to thereby get a respective representation of the metabolic activity of individual biological entities in response to exposure of the chemical agent. The actual response of the biological entities in the microwells 20 to the chemical agent can then be determined based on the control / reference and experimental measurements. Figs. 10-13 illustrate an embodiment of the monitoring device 1 comprising an array or matrix 21 of microwells 20. In this embodiment, the channel plate 30 comprises a single fluid channel 40 dimensioned to be in fluid connection with all microwells 20 in the microwell plate 10 in the loading position as schematically shown in Figs. 12A, 13. In an alternative embodiment, the single fluid channel 40 of the channel plate 30 could be dimensioned to merely be aligned with and in fluid connection with a subset of the microwells 20 in the array or matrix 21 in the loading position. In such a case, the loading position could include multiple loading positions, in which the channel plate 30 is in fluid connection with different subsets of microwells 20. For instance, assume that the array of matrix 21 of microwells 20 includes 2N columns of M microwells 20 and that the width of the single fluid channel 40 matches or is slightly larger than the width of N columns of microwells 20. Then the relative movement between the microwell plate 10 and the channel plate 30 could move the single fluid channel 40 to first be in fluid connection with a first subset of NxM microwells 20 in a first loading position and then move the single fluid channel 40 to be in fluid connection with the second and remaining subset of NxM microwells 20 in a second loading position. Such an embodiment enables exposing biological entities in different subset of microwells 20 to different chemical agents during the same measurement session of the monitoring device 1.

[0051] Figs. 14-16 illustrate another embodiment of the monitoring device 1 comprising multiple, such as two, subsets 21 , 23, such as arrays or matrices, of microwells 20. In this embodiment, the channel plate 30 comprises multiple fluid channels 40, 42, such as one fluid channel 40, 42 per subset 21 , 23 of microwells 20. As is shown in Fig. 16A, in the loading position, a first fluid channel 40 is in fluid connection with a first subset 21 of microwells 20, whereas a second fluid channel 42 is in fluid connection with a second subset 23 of microwells 20. In the monitoring position, see Fig. 16B, the relative movement between the microwell plate 10 and the channel plate 30 moves the fluid channels 40, 42 away from the subsets 21 , 23 of microwells 20 so that the channel plate 30 instead closes the microwells 20. The embodiments are not limited to using merely two fluid channels 40, 42 and two subsets 21 , 23 of microwells 20. For instance, the plurality of microwells 20 in the microwell plate 10 could be arranged as one, two, three or more subsets 21 , 23, such as arrays or matrices, of microwells 20 and the channel plate 30 could include one, two, three or more fluid channels 40, 42.

[0052] Hence, in an embodiment, the channel plate 30 comprises multiple fluid channels 40, 42 arranged in the first main surface 34 of the channel plate 30. Each fluid channel 40, 42 of the multiple fluid channels 40, 42 is in fluid connection with a respective fluid port 11A, 11 B or a common fluid port 11 and a respective fluid output port 13A, 13B or a common fluid output port 13 in the loading position. Further, each fluid channel 40, 42 of the multiple fluid channels 40, 42 is in fluid connection with a respective subset 21 , 23 of the plurality of microwells 20 in the loading position.

[0053] The fluid channel(s) 40, 42 of the channel plate 30 is(are) in fluid connection with a fluid input port 11 ; 11 A, 11 B and a fluid output port 13; 13A, 13B. These ports 11, 13; 11 A, 11 B, 13A, 13B are used to flow a fluid through the fluid channel(s) 40, 42 from the fluid input port(s) 11 ; 11 A, 11 B, through the fluid channel(s) 40, 42 and out through the fluid output port(s) 13; 13A, 13B. In the case of multiple fluid channels 40, 42 in the channel plate 30 then each such fluid channel 40, 42 may be in fluid connection with a respective fluid input port 11 A, 11 B and a respective fluid output port 13A, 13B. Alternatively, at least two of the multiple fluid channels 40, 42 may share and have a common fluid input port 11 and / or a common fluid output port 13. Thus, the embodiments encompass, each fluid channel 40, 42 being, in the loading position, in fluid connection with a respective fluid input port 11 A, 11 B and a respective fluid output port 13A, 13B; in fluid connection with a respective fluid input port 1 1A, 11 B and a common fluid output port 13; in fluid connection with a common fluid input port 11 and a respective fluid output port 13A, 13B; or in fluid connection with a common fluid input port 11 and a common fluid output port 13.

[0054] In an embodiment, the microwell plate 10 comprises the fluid input port(s) 11 ; 11 A, 11 B in a first end surface 15 and the fluid output port(s) 13; 13A, 13B in a second, opposite end surface 17. The fluid input port(s) 11 ; 11 A, 11 B is(are) then preferably in fluid connection with a first respective opening 16; 16A 16B in the first main surface 12 of the microwell plate 10. Correspondingly, the fluid output port(s) 13; 13A, 13B is(are) preferably in fluid connection with a respective second opening 18; 18A, 18B in the first main surface 12 of the microwell plate 10. In such an embodiment, the at least one fluid channel 40, 42 is in fluid connection with the respective first opening 16; 16A, 16B and the respective second opening 18; 18A, 18B in the loading position.

[0055] Fig. 13 schematically shows the monitoring device 1 in the loading position. As is seen in the figure, the fluid channel 40 is in fluid connection with the first opening 16 and the second opening 18 in the first main surface 12 of the microwell plate 10. As a consequence, a fluid entering the fluid input port 11 will flow through the first opening 16 and into the fluid channel 40 and then further out through the second opening 18 and the fluid output port 13. In such an embodiment, any tubing for the fluid could be connected to the fluid input port 11 in the first end surface 15 of the microwell plate 10 and the fluid output port 13 in the second end surface 17 of the microwell plate 10. In an embodiment, the microwell plate 10 comprises at least one matrix or array 21 , 23 of microwells 20 arranged in the first main surface 12 of the microwell plate 10. For instance, the plurality of microwells 20 could be arranged as one or more matrices or arrays 21 , 23 in the first main surface 12 of the microwell plate 10. As an example, the microwell plate 10 can include N columns and M rows of microwells 20, i.e., an NxM matrix of microwells 20, wherein N and M represent positive integer numbers and at least one of N and M, preferably both of N and M, is a positive integer number larger than 1 . If the microwells 20 are divided into multiple subsets 21 , 23, such as multiple matrices or arrays, then these subsets 21 , 23, such as matrices or arrays, may contain the same number of microwells 20, such as an NxM matrix of microwells 20 each, or different numbers of microwells 20, such as an NkxMk matrix of microwells 20, wherein each Nk and Mk is a positive integer number of which at least one, preferably both, is larger than one, and / r=1...Q and Q represents the number of subsets 21 , 23, such as matrices or arrays, in the multi-channel plate 10.

[0056] The microwell plate 10 and the channel plate 30 are arranged so that the first main surface 12 of the microwell plate 10 faces the first main surface 34 of the channel plate 30. In an embodiment, the first main surface 12 of the microwell plate 10 is in direct physical contact with the first main surface 34 of the channel plate 30. In such an embodiment, the first main surfaces 12, 23 slide on each other during the relative movement between the microwell plate 10 and the channel plate 30.

[0057] In another embodiment, a gas-impermeable sliding polymer film or coating 50 could be attached to the first main surface 34 of the channel plate 30, to the first main surface 12 of the microwell plate 10, or a first gas-impermeable sliding polymer film or coating 50 is attached to the first main surface 34 of the channel plate 30 and a gas-impermeable second sliding polymer film or coating is attached to the first main surface 12 of the microwell plate 10.

[0058] In a preferred embodiment, a gas-impermeable sliding polymer film or coating 50 is attached to the first main surface 34 of the channel plate 30. In such an embodiment, the gas-impermeable sliding polymer film or coating 50 is in slidable contact with the first main surface 12 of the microwell plate 10.

[0059] Such a gas-impermeable sliding polymer film or coating 50 could be used to facilitate sliding of the channel plate 30 relative to the microwell plate 10 and / or vice versa. Thus, the gas-impermeable sliding polymer film or coating 50 presents a lower coefficient of friction as compared to the first main surface 34 of the channel plate 30 and thereby makes it easier to perform the relative movement between the channel plate 30 and the microwell plate 10 between the loading position and the monitoring position.

[0060] In an embodiment, the gas-impermeable sliding polymer film or coating 50 not only facilitates the relative movement between the channel plate 30 and the microwell plate 10 but is also fluid tight and forms a fluid tight seal of individual microwells 20 in the at least a subset 21 , 23 of the plurality of microwells 20 in the monitoring position as more clearly seen in Figs. 12B, 16B. In such an embodiment, the gas- impermeable sliding polymer film or coating 50 could be regarded as a fluid-impermeable polymer film or coating 50. The gas-impermeable or fluid-impermeable sliding polymer film or coating 50 thereby prevents or at least restricts any cross-talk between adjacent microwells 20 in the monitoring position and thereby prevents or at least restricts agents, and in particular the analyte, to which the optode compound is sensitive, to leak from one microwell 20 into an adjacent microwell 20.

[0061] The gas-impermeable or fluid-impermeable sliding polymer film or coating 50 could be in the form of a single gas-impermeable or fluid-impermeable polymer film or coating 50 or a stack of multiple polymer films or coatings 50. For instance, a fluid-impermeable sliding polymer film or coating 50 could comprise a liquid-tight film or coating of a first polymer attached to the first main surface 34 of the channel plate 30 and a gas-impermeable film or coating of a second polymer attached to the liquid-tight film or coating. Hence, in the case of a stack of multiple polymer films or coatings, polymer films or coatings with different characteristics such as in terms of liquid-impermeability, fluid-impermeability, friction, etc., could be used.

[0062] An example of the first polymer that could be used for the liquid-tight film or coating is polydimethylsiloxane (PMDS) and an example of the second polymer that could be used for the gas- impermeable film or coating is polyethylene terephthalate (PET or PETE).

[0063] The gas-impermeable sliding polymer film or coating 50 of thereby has multiple benefits to the monitoring device 1. The gas-impermeable sliding polymer film or coating 50 facilitates the relative movement between the channel plate 30 and the microwell plate 10 between the loading position and the monitoring position by presenting a lower coefficient of friction as compared to the first main surface 34 of the channel plate 30. Further, the gas-impermeable sliding polymer film or coating 50 prevents or restricts crosstalk or contamination between the microwells 20 of the microwell plate 10. For instance, the analyte-sensitive optode compound of the optode composition 25 could be sensitive to and respond to an analyte in a gaseous state, i.e. , being a gas. In such an embodiment, the gas-impermeable sliding polymer film or coating 50 inhibits gas produced and / or consumed by a biological entity in one microwell 20 of the microwell plate 10 from entering another microwell 20 of the microwell plate 10. The gas-impermeable sliding polymer film or coating 50 thereby prevents, or at least inhibits or restricts, contamination of the analyte between the microwells 20 of the microwell plate.

[0064] This embodiment is a significant advantage over the SlipChip device as disclosed in WO 2010 / 111265 A1. The SlipChip device is assembled under a perfluorinated oil (FC-40, also known as 3MTMFluorinert™), which resulted in that all channels and surfaces of the SlipChip device were wet by FC-40. The space between the sliding surfaces of the SlipChip device was thereby filled with FC-40.

[0065] Experimental data as presented herein show that such a usage of a FC-40 coating between the base and plate of the SlipChip device is not compatible with isolating the wells from each other but rather leads to crosstalk and contamination between adjacent wells.

[0066] This should be compared to an embodiment of the invention were the microwells 20 in the microwell plate 20 are isolated from each other by the gas-impermeable polymer film or coating 50, see Fig. 3.

[0067] Further, the SlipChip device as disclosed in WO 2010 / 111265 A1 mentions usage of FEP as a coating material to prevent wetting of areas of the SlipChip device beyond the wells or as a lubricating material. Experimental data as presented herein, see Fig. 18, shows that such a FEP coating is not gas- impermeable and can therefore not be used as a gas-impermeable sliding polymer film or coating.

[0068] The microwells 20 of the microwell plate 10 could be of any shape and dimensions suitable to house at least one, preferably one, biological entity. Thus, the dimensions of the microwells 20 could be varied depending on the particular biological entities to be monitored by the monitoring device 1. In a typical example, the microwells 20 are in the form of cylinders with a bottom or base 24 and an inner cylinder or curved wall 22, see Fig. 10.

[0069] In an embodiment, the microwells 20 of the microwell plate 10 are dimensioned to house, on average, at most one biological entity each. Thus, the dimensions of the microwells 20 are then adapted to the particular biological entities to be tested and the dimensions of these biological entities. Such an embodiment reduces the risk of having multiple biological entities captured in a same microwell 20.

[0070] Biological entity as used herein, also referred to as biological unit in the art, include cells, organoids, spheroids, and other two- or three-dimensional cultures comprising multiple cells together constituting an organoid or spheroid. Hence, in an embodiment, biological entity is selected from the group consisting of a cell, an organoid and a spheroid, preferably selected from the group consisting of a cell and an organoid.

[0071] As an example, the biological entity is a cell. The monitoring device 1 could then be regarded as a singlecell monitoring device comprising a microwell plate 10 comprising microwells 20 dimensioned to hold a respective cell.

[0072] In another example, the biological entity is an organoid. In such an embodiment, the monitoring device 1 could be regarded as a single-organoid monitoring device 1 comprising a microwell plate 10 comprising microwells 20 dimensioned to hold a response organoid.

[0073] An organoid is a miniaturized and simplified version of an organ produced in vitro in three dimensions that mimics the key functional, structural, and biological complexity of that organ. It is typically derived from one or a few cells from a tissue, embryonic stem cells, or induced pluripotent stem cells, which can self-organize in three-dimensional culture owing to their self-renewal and differentiation capacities.

[0074] In a further example, the biological entity is a spheroid. In such an embodiment, the monitoring device 1 could be regarded as a single-spheroid monitoring device 1 comprising a microwell plate 10 comprising microwells 20 dimensioned to hold a response spheroid.

[0075] A spheroid is a three-dimensional cluster of cells that self-assemble into a roughly spherical shape when cultured in non-adherent conditions. They are commonly used in research to better mimic the in vivo environment compared to traditional two-dimensional cell cultures.

[0076] As mentioned in the foregoing, the microwells 20 of the microwell plate 10 are preferably dimensioned to house, on average, at most one biological entity each. The size of the microwells 20 is then dependent on the particular type of biological entities to monitor. For instance, organoids and spheroids could have a diameter of 300 pm or more, whereas a single cell typically has a diameter of about 50 pm.

[0077] The optode composition 25 is present in the microwells 20. For instance, the optode composition 25 could be deposited on at least a portion of the surfaces 22, 24 of the microwells 20. For instance, the optode composition 25 could be deposited on the bottom or base 24, or at least a portion thereof, and / or on the inner cylinder or curved wall 22, or at least a portion thereof. The optode composition 25 comprises the analyte-sensitive optode compound that is sensitive to and responds to the analyte. The analyte-sensitive optode compound thereby generates an optical signal in response to the analyte. In an embodiment, the optode composition 25 also comprises an analyteinsensitive reference compound capable of generating a detectable optical reference signal. This analyteinsensitive reference compound is not sensitive to the analyte and the detectable optical reference signal generated by the analyte-insensitive reference compound is thereby not representative of the amount or concentration of the analyte. However, the detectable optical signal generated by the analyte-sensitive optode compound is preferably representative of the amount or concentration of the analyte.

[0078] The analyte-insensitive reference compound is typically in the form of a dye and a particular example is 3-(5-chlorobenzoxazol-2-yl)-7-diethylaminocoumarin.

[0079] In an embodiment, the analyte-sensitive optode compound is a gas-sensitive optode compound, i.e., an analyte-sensitive optode compound sensitive to an analyte in gaseous state or form, i.e., a gas analyte.

[0080] The analyte-sensitive optode compound is preferably selected from the group consisting of an oxygensensitive optode compound, a pH-sensitive optode compound, and a carbon dioxide-sensitive optode compound.

[0081] An oxygen-sensitive optode compound generates a detectable optical signal in response to the presence of oxygen (O2). Illustrative, but non-limiting, example of such 02-sensitive optode compounds include Pt(ll) meso-tetra(pentafluoropheynyl)porphine (PtTFPP) and platinum(ll) benzoporphyrin (Staudinger et al., Limnology and Oceanography Methods (2018) 16: 459-473).

[0082] A pH-sensitive optode compound is sensitive to pH changes and thereby generates a detectable optical signal representative of the pH. Illustrative, but non-limiting, example of such pH-sensitive optode compounds include a BF2-chelated tetraarylazadipyrromethene (Jokic et al., Analytical Chemistry (2012) 84(15): 6723-6730) and aza-BODIPY dyes (Strobl et al., Analyst (2015) 140(21): 7150-7153), such as 4,4'-(5, 5-difl uoro- 1 , 9-dip henyl-5H-4X4, 5X4-dipyrrolo-[1 ,2-c:2',1'-f|[1 , 3, 5, 2]triazabori ni ne-3, 7- diyl)diphenol.

[0083] A carbon dioxide-sensitive optode compound generates a detectable optical signal in response to the presence of carbon dioxide (CO2). An illustrative, but non-limiting, example of such CO2-sensitive optode compounds is SF-CD1 R from Presens (Wiorek et al., ACS Sensors (2023) 8: 2843-2851). In an embodiment, the analyte-sensitive optode compound is selected from the group consisting of Pt(ll) meso-tetra(pentafluoropheynyl)porphine, a BF2-chelated tetraarylazadipyrromethene, and 4,4'-(5,5- difluoro-1 ,9-diphenyl-5H-4X4,5X4-dipyrrolo-[1 , 2-c: 2', 1 '- / ] [1 , 3, 5, 2]triazabori ni ne-3, 7-diyl)dip henol .

[0084] In an embodiment, the optode composition 25 comprises a matrix material for the analyte-sensitive optode compound and the optional analyte-insensitive reference compound. An example of such a matrix material is a polymer material, such as polystyrene (PS).

[0085] In an embodiment, the optode composition 25 may comprise an adherence promoting agent that promotes adherence of the optode composition 25 to the inner surfaces 22, 24 of the microwells 20. For instance, if the microwell plate 10 is made of glass then silicone rubber could be used as adherence promoting agent.

[0086] Hence, in an embodiment, the optode composition 25 also comprises polystyrene and preferably silicone rubber.

[0087] In an embodiment, at least the portion of the microwell plate 10 comprising the plurality of microwells 20 is optically transparent. In such a case, the detectable optical signals generated by the analyte-sensitive optode compound in the optode composition 25 in the microwells 20 and the optional detectable optical reference signals generated by the analyte-insensitive reference compound if included in the optode composition 25 can be detected and measured through this optically transparent portion of the microwell plate 10. In a preferred embodiment, the complete microwell plate 10 is made of an optically transparent material.

[0088] In an alternative, or additional embodiment, at least a portion of the channel plate 30 aligned with the at least a subset 21 , 23 of the plurality of microwells 20 is optically transparent. In such a case, the detectable optical signals generated by the analyte-sensitive optode compound in the optode composition 25 in the microwells 20 and the optional detectable optical reference signals generated by the analyteinsensitive reference compound if included in the optode composition 25 can be detected and measured through this optically transparent portion of the channel plate 30. In a preferred embodiment, the complete channel plate 30 is made of an optically transparent material. Illustrative, but non-limiting, examples of materials for the microwell plate 10 and the channel plate 30 include optically transparent polymers, optically transparent plastics and optically transparent glass.

[0089] In an embodiment, the microwell plate 10 is made of an optically transparent material, preferably glass, and more preferably borosilicate glass.

[0090] In an embodiment, the channel plate 30 is made of an optically transparent material, preferably glass, and more preferably fused silica glass.

[0091] In an embodiment, the microwell plate 10 and the channel plate 30 are made of a gas-impermeable material.

[0092] The present invention also relates to a monitoring system 100, see Fig. 1 D. The monitoring system 100 comprises a monitoring device 1 according to the invention, a light system 110 and a detector 120. The light system 110 comprises a light source 112 and is configured to direct input light 111 into the at least a subset 21 , 23 of the plurality of microwells 20. The detector 120 is then configured to detect output light 113, 115 emitted by the optode composition 25 in response to the input light 11 and based on the analyte.

[0093] The light source 112 of the monitoring system 100 is configured to generate input light 111 that is directed into the at least subset 21 , 23 of the plurality of microwells 20. The particular wavelength or wavelength range of the input light 111 generated by the light source 112 is preferably selected at least partly based on the particular analyte-sensitive optode compound and optional analyte-insensitive reference compound of the optide composition 25 present in the microwells 20. The wavelength or wavelength interval may, for instance, be within the visible spectrum (400-700 nm), ultraviolet (UV) spectrum (10-400 nm) and / or infrared (IR) spectrum (750 nm - 1 mm).

[0094] The light source 112 could be any light source capable of generating input light 111 of the desired wavelength or wavelength interval. Illustrative, but non-limiting, examples of such light sources include UV lamps, lasers, IR lamps, and lamps producing visible light.

[0095] The output light 113, 115 generated by the optode composition 25 present in the microwells 20, and in particular by the analyte-sensitive optode compound and the optional analyte-insensitive reference compound in response to the input light 111 is detected by the detector 120. The output light 113, 1 15 could be of a single wavelength, of multiple wavelengths, of a single wavelength interval or of multiple wavelength intervals. For instance, the analyte-sensitive optode compound could generate output light 113 at a first wavelength or first wavelength interval in response to the input light 111 and where this output light 113 is representative of the amount or concentration of the analyte in the respective microwells 20. Similarly, the analyte-specific compound could generate changes in the luminescence over time in response to the concentration of the to-be-detected analyte. Correspondingly, the analyteinsensitive reference compound, if present, could generate output light 115 at a second wavelength or second wavelength interval in response to the input light 111 and where this output light 115 is not representative of the amount or concentration of the analyte in the respective microwells 20.

[0096] The detector 120 could be any detector capable of detecting the output light 113, 115 produced by the optode composition 25 in the microwells 20. Illustrative, but non-limiting, examples of such detectors include cameras, such as RGB cameras, UV cameras, or IR cameras, photosensors, such as metalsemiconductor-metal (MSM) photodetectors, photodiodes, avalanche photodiodes, phototransistors, charge-coupled devices, CMOS image sensors, or photomultiplier tubes.

[0097] In an embodiment, the light system 110 comprises the above-mentioned light source 112 and a light guide to direct the input light 11 1 generated by the light source 112 into the at least a subset 21 , 23 of the plurality of microwells 20. Alternatively, or in addition, the light system 110 comprises a dichroic mirror arranged to reflect light from the light source 112 into the at least a subset 21 , 23 of the plurality of microwells 20. The dichroic mirror is then arranged to reflected light from the light source 112, i.e., the input light 111 , within the desired wavelength interval or desired wavelength for the input light 111. The dichroic mirror is then able to transmit, i.e., not reflect, the output light 113, 115 from the monitoring device 1 but rather allow such output light 113, 115 to pass through the dichroic mirror with no or almost no absorption of the wavelength(s) or wavelength interval(s) of the output light 113, 115.

[0098] The monitoring system 100 may also include an emission filter arranged in the light path from the monitoring device 1 and the detector 120 and preferably between the dichroic mirror and the detector 120. The emission filter is preferably configured to allow the desirable wavelength(s) or wavelength interval(s) of the output light 113, 115 to reach the detector 120 while blocking unwanted traces of the input light 111 from the light source 112.

[0099] The monitoring system 100 may optionally comprise an objective arranged to focus the light rays of the input light 111 into the illuminated microwell(s) 20 in the monitoring device 1 and / or focus the light rays of the output light 113, 115 into the detector 120. In an embodiment, the monitoring system 100 comprises a device holder 130, see Fig. 1 A, comprising at least one micrometer screw 132 arranged to move the channel plate 30 relative to the microwell plate 10, or vice versa. In this embodiment, one or multiple, preferably two, micrometer screws 132 are arranged to be connected to the channel plate 30 or the microwell plate 10. Screwing the micrometer screws 132 clockwise or counter-clockwise then moves the channel plate 30 relative to the microwell plate 10 or moves the microwell plate 10 relative to the channel plate 30. In an alternative embodiment, a first micrometer screw 132 or a first pair of micrometer screws 132 is or are arranged to move the channel plate 30, whereas a second micrometer screw or a second pair of micrometer screws 132 is or are arranged to move the microwell plate 10. In these embodiments, the micrometer screw(s) could be manually operated or driven by a motor to move the channel plate 30 and the microwell plate 10 relative to each other between the loading position and the monitoring position.

[0100] Fig. 2 illustrates an example of components of the device holder 130 and the micrometer screw(s) 132 according to an embodiment.

[0101] Another aspect of the invention relates to a method of monitoring biological entities, see Fig. 7. The method comprises loading biological entities into microwells 20 of the monitoring device 1 of the invention. The method also comprises moving the channel plate 30 relative to the microwell plate 10, or vice versa, to the monitoring position. Input light 111 is then directed into the at least a subset 21 , 23 of the plurality of microwells 20 and output light 113, 115 is detected, for each microwell 20 of the at least a subset 21 , 23 of the plurality of microwells 20 comprising at least one biological entity, from the microwell 20. A reference value is generated, for each microwell 20 of the at least a subset 21 , 23 of the plurality of microwells 20 comprising at least one biological entity, based on the detected output light 113, 115.

[0102] The channel plate 30 is then moved relative to the microwell plate 10, or vice versa, to the loading position and a fluid is flown through the at least one fluid channel 40, 42 in the loading position. The channel plate 30 is thereafter moved relative to the microwell plate 10, or vice versa, to the monitoring position. Input light 111 is then directed into the at least a subset 21 , 23 of the plurality of microwells 20 and output light 113, 115 is detected, for each microwell 20 of the at least a subset 21 , 23 of the plurality of microwells 20 comprising at least one biological entity , from the microwell 20. A measurement value is generated, for each microwell 20 of the at least a subset 21 , 23 of the plurality of microwells 20 comprising at least one biological entity , based on the detected output light 113, 115. An analyte response of biological entities to the fluid is determined based on the measurement value and the reference value determined for each microwell 20 of the at least a subset 21 , 23 of the plurality of microwells 20 comprising at least one biological entity.

[0103] The method, thus, involves loading biological entities into the microwells 20 of the microwell plate 10, preferably at least one biological entity and more preferably only one biological entity per microwell 20. The microwell plate 10 and the channel plate 30 are then moved relative to each other into the measuring position seen as step 2 in Fig. 7. At this point input light 111 is directed to the microwells 20 and the output light 113, 115 therefrom is detected.

[0104] In an embodiment, an initial monitoring of the microwells 20 in the microwell plate 10 is first done following cell loading but prior to detection of the output light 113, 115. This initial monitoring could be in the form of a visual inspection of the microwells 20, such as using a microscope, or by taking an image or picture of the microwells 20 in order to identify the microwells 20 of the microwell plate 10 comprising at least one biological entity, and optionally identifying those microwells 20 containing a single biological entity each. For instance, if the microwells 20 are arranged as one or multiple matrices or arrays 21 , 23 of microwells 20 then the coordinates of the microwells 20 containing at least one biological entity, or containing only one biological entity, are identified from the initial monitoring. Fig. 8C illustrates at 0 min the identification of a microwell 20 comprising a single biological entity.

[0105] A reference or control light measurement is then conducted prior to exposing the biological entities captured in the microwells 20 to the fluid. Thus, the output light 113, 115 generated by the optode composition 25 in the microwells 20 in response to the input light 111 is detected and used to generate a respective reference value for at least each microwell 20 comprising at least one biological entity or a single biological entity in the at least a subset 21, 23 of the plurality of microwells 20.

[0106] The microwell plate 10 and the channel plate 30 are then moved relative to each other into the loading position, at which the at least one fluid channel 40, 42 is in fluid connection with the at least a subset 21 , 23 of the plurality of microwells 20. A fluid is then flowing through the at least one fluid channel 40, 42 and the biological entities captured in the microwells 20 are thereby exposed to the fluid. The fluid could be a gas or a liquid. The gas or liquid could, for instance, contain a carrier gas or liquid, in which a chemical agent to be tested is mixed, dispersed, or dissolved. Alternatively, the gas or liquid as such could the chemical agent in gaseous form or liquid form. The microwell plate 10 and the channel plate 30 are then moved relative to each other into the measuring position, at which the channel plate 30 closes the microwells 20, see step 3 of Fig. 7. A light measurement is then conducted following exposing the biological entities captured in the microwells 20 to the fluid. Thus, the output light 113, 115 generated by the optode composition 25 in the microwells 20 in response to the input light 111 is detected and used to generate a respective measurement value for at least each microwell 20 comprising at least one biological entity or a single biological entity in the at least a subset 21 , 23 of the plurality of microwells 20.

[0107] A respective analyte response is then determined for each biological entity / microwell 20 based on the measurement value and the reference value generated for the particular biological entity / microwell 20.

[0108] As mentioned in the foregoing, during loading of biological entities some microwells 20 could be empty, other microwells 20 could contain more than one biological entity each, whereas further microwells 20 contain only a single biological entity cell each. If the analyte response is to be determined per biological entity then the above-mentioned initial monitoring could be used to identify those microwells 20 that contain a single biological entity each. In such a case, this initial monitoring could be used to filter out empty microwells 20 or microwells containing more than one biological entity each. This “filtering” could be done upon generation of the reference and measurement values, i.e., only generating reference and measurement values for those microwells 20 identified as containing a single biological entity each. Alternatively, the “filtering” could be done upon determining the analyte responses so that the analyte response is only determined for those microwells 20 identified as containing a single biological entity each. As another alternatively, an analyte response could be determined for each microwell 20 and then the “filtering” is done afterwards by identifying those analyte responses that are determined for microwells 20 containing a single biological entity each.

[0109] The loading of biological entities into the microwells 20 of the microwell plate 10 can be performed according to various embodiments.

[0110] In an embodiment, a suspension of biological entities is added to microwells 20 of the monitoring device 1 in an open position, see Fig. 12C, in which the channel plate 30 is not covering the at least a subset 21 , 23 of the plurality of microwells 20 in the microwell plate 10. The channel plate 30 is then moved relative to the microwell plate 10, or vice versa, to the loading position, see Fig. 12A, and a culture medium is flown through the at least one fluid channel 40, 42 in the loading position. This embodiment thereby involves opening the microwells 20 in the microwell plate 10 by moving the channel plate 30 away from the at least a subset 21, 23 of the plurality of microwells 20, or by moving the microwell plate 10 away from the channel plate 30. In this open position, the channel plate 30 does not cover the microwells 20, which are thereby open and accessible for addition of the suspension of biological entities. The suspension could, for instance, be pipetted or poured into the microwells 20. The microwell plate 10 and the channel plate 30 are then moved relative to each other to get into the loading position, in which the at least one fluid channel 40, 42 is in fluid connection with the at least a subset 21, 23 of the plurality of microwells 20. A culture medium can then be flown through the at least one fluid channel 40, 42 to remove any surplus biological entities, while promoting deposition of the biological entities from the suspension into the bottoms 24 of the microwells 20.

[0111] In another embodiment, a suspension of biological entities is flown through the at least one fluid channel 40, 42 in the loading position followed by flowing a culture medium through the at least one fluid channel 40, 42 in the loading position.

[0112] In this embodiment, the suspension is initially flown through the at least one fluid channel 40, 42 to thereby enter the microwells 20 and allow biological entities in the suspension to enter the microwells 20. The suspension is then replaced by a culture medium to remove any surplus biological entities, while promoting deposition of biological entities from the suspension into the bottoms 24 of the microwells 20.

[0113] The suspension of biological entities used in the above-mentioned embodiments can be any suspension comprising biological entities to be tested in the monitoring device 1. As an illustrative, but non-limiting, example the suspension could be a culture medium comprising biological entities. The same culture medium, or another culture medium, could then be flown through the at least one fluid channel 40, 42 in the loading position.

[0114] In an embodiment, the culture medium is flown through the at least one fluid channel 40, 42 in the loading position at a first fluid pressure. The fluid pressure is then reduced to a second fluid pressure that is lower than the first fluid pressure while continuing to flow the culture medium through the at least one fluid channel 40, 42 in the loading position.

[0115] For instance, a first pressure, such as 30 mbar as an illustrative but non-limiting example, can be used for the culture medium to remove cells outside of microwells 20 and subsequently the pressure is reduced over a time period to a second pressure, for instance reduced over 1 min to a final pressure of 10 mbar as an illustrative but non-limiting example. This procedure helps retaining biological entities within microwells 20 while effectively washing away excess biological entities outside of the microwells 20.

[0116] In an embodiment, the optode composition 25 comprises the analyte-insensitive reference compound capable of generating a detectable optical reference signal in addition to the analyte-sensitive optode compound. In such an embodiment, the method comprises detecting, for each microwell 20 of the at least a subset 21 , 23 of the plurality of microwells 20 comprising at least one biological entity, output light 113 emitted by the analyte-sensitive optode compound and reference output light 115 emitted by the analyteinsensitive reference compound from the microwell 20. This embodiment also comprises generating, for each microwell 20 of the at least a subset 21 , 23 of the plurality of microwells 20 comprising at least one biological entity, the reference value based on the detected output light 113 and the detected reference output light 115.

[0117] The channel plate 30 is then moved relative to the microwell plate 10, or vice versa, to the loading position and a fluid is flown through the at least one fluid channel 40, 42 in the loading position. The channel plate 30 is thereafter moved relative to the microwell plate 10, or vice versa, back to the monitoring position.

[0118] This embodiment also comprises detecting, for each microwell 20 of the at least a subset 21 , 23 of the plurality of microwells 20 comprising at least one biological entity, output light 113 emitted by the analytesensitive optode compound and reference output light 115 emitted by the analyte-insensitive reference compound from the microwell 20 and generating, for each microwell 20 of the at least a subset 21 , 23 of the plurality of microwells 20 comprising at least one biological entity, the measurement value based on the detected output light 113 and the detected reference output light 115.

[0119] Thus, the reference and measurement values are, in this embodiment, determined not only based on the output light 113 generated by the analyte-sensitive optode compound based on the input light 111 and representative of the amount or concentration of the analyte in the particular microwell 20 but also based on the reference output light 115 generated by the analyte-insensitive reference compound based on the input light 111.

[0120] For instance, the reference value and the measurement value could be determined based on a ratio between a characteristic of the output light 113 and the characteristic of the reference output light 115. For instance, the reference value (r0) and the measurement value (r) could be calculated as the ratio of the intensity of the output light 113 (Z10, / and the intensity of the reference output light 115 (Z2,0>

[0121] In an embodiment, the analyte response of cells to the fluid is determined based on a ratio between the measurement value and the reference value determined for each microwell 20 of the at least a subset 21 , 23 of the plurality of microwells 20. For instance, the analyte response (A) is a function ( ( )) of the ratio between the measurement value (r) and the reference value (r0), i.e. , A = f ( — ) or A =

[0122] / (?)■

[0123] As an example, determining the analyte response of cell to the fluid could involve determining a concentration of the analyte [A] based on the equation: wherein r represents the measurement value, r0represents the reference value, and ksvrepresents the Stern-Volmer constant.

[0124] As an example, the detector 120 could be in the form of a red, green, blue (RGB) camera that is arranged to take RGB images of the at least a subset 21 , 23 of the plurality of microwells 20. To process the obtained RGB images, the pixel-wise ratiometric signals (r) of the emission of light at a first wavelength or first wavelength interval attributable to the analyte-sensitive optode compound and the emission of light at a second wavelength or a second wavelength interval emanating from the analyte-insensitive reference compound are extracted from the RGB images and subsequently analyzed through the application of the Stern-Volmer relationship:

[0125] Here r = - signifies the ratio of luminescence intensities ( / ) between the first wavelength or wavelength interval and the second wavelength or wavelength interval in the presence of the analyte, being a function of analyte concentration [A]; r0denotes ratio between luminescence intensities between the first wavelength or wavelength interval and the second wavelength or wavelength interval in the absence of the analyte; ksvrepresents the Stern-Volmer constant.

[0126] An advantage of the monitoring device 1 of the invention is that the monitored biological entities can be retrieved from the microwells 20 following the light measurements. Thus, the analyzed biological entities could be removed from the monitoring device 1 for further processing or usage. In such an embodiment, the method also comprises moving the channel plate 30 relative to the microwell plate 10, or vice versa, to an open position, see Fig. 12C, in which the channel plate 30 is not covering the at least a subset 21 , 23 of the plurality of microwells 20 in the microwell plate 30. The method also comprises, in this embodiment, retrieving biological entities from microwells 20 of the at least a subset 21 , 23 of the plurality of microwells 20.

[0127] The present invention also relates to a method of producing a monitoring device 1 . The method comprises generating, in a first substrate, a plurality of microwells 20 dimensioned to hold a respective cell to form a microwell plate 10 comprising the plurality of microwells 20 in a first main surface 14 of the microwell plate 10. The method also comprises generating, in a second substrate, at least one fluid channel 40, 42 to form a channel plate 30 comprising the at least one fluid channel 40, 42 in a first main surface 34 of the channel plate 30. The method further comprises depositing an optode composition 25 in the plurality of microwells 20. The optode composition 25 comprises an analyte-sensitive optode compound generating a detectable optical signal in response to an analyte. The method additionally comprises slidably arranging the channel plate 30 and the microwell plate 10 together with the first main surface 34 of the channel plate 30 facing the first main surface 12 of the microwell plate 10 so that the channel plate 30 and the microwell plate 10 are movable relative to each other between a loading position, in which the at least one fluid channel 40, 42 is in fluid connection with at least a subset 21 , 23 of the plurality of microwells 20, and a monitoring position, in which the channel plate 30 closes the at least a subset 21 , 23 of the plurality of microwells 20. At least the portion of the microwell plate 10 comprising the plurality of microwells 20 is optically transparent and / or at least the portion of the channel plate 30 aligned with the at least a subset 21 , 23 of the plurality of microwells 20 in the monitoring position is optically transparent.

[0128] Fig. 4A schematically illustrates an embodiment of generating the microwell plate 10 by dry etching the microwells 20 in a first main surface of a borosilicate glass substrate and Fig. 4B schematically illustrates an embodiment of generating the channel plate 30 by wet etching the at least one fluid channel 40, 42 in a first main surface of a fused silica glass substrate, which is described in more detail in the Example section. Fig. 5 schematically illustrates an embodiment of deposition of the optode composition 25 in the microwells 20 of the microwell plate 10.

[0129] In an embodiment, the method further comprises attaching a gas-impermeable sliding polymer film or coating 50 attached to the first main surface 34 of the channel plate 30. The sliding polymer film or coating 50 is in slidable contact with the first main surface 12 of the microwell plate 10.

[0130] In an embodiment, attaching the gas-impermeable sliding polymer film or coating 50 comprises attaching a liquid-tight film or coating of a first polymer, preferably polydimethylsiloxane, to the first main surface 34 of the channel plate 30. This embodiment also comprises attaching a gas-impermeable film or coating of second polymer, preferably polyethylene terephthalate, to the liquid-tight film or coating.

[0131] EXAMPLES

[0132] EXAMPLE 1

[0133] In disciplines like toxicology and pharmacology, oxygen (O2) respiration is a universal metric for evaluating the effects of chemicals across various model systems, including mammalian and microalgal cells. However, for these cells the common practice is to segregate populations into control and exposure groups, which assumes direct equivalence in their responses and does not take into account cellular heterogeneity. This lack of resolution impedes our ability to precisely investigate differences among experimental groups in rare samples. To overcome this barrier, SlipO2Chip™, an innovative microfluidic platform tailored for precisely quantifying single-cell O2 respiration in the coordinated absence and presence of chemical solutes was developed in this example. Constructed in glass, SlipC>2Chip™ comprised a wet-etched channel plate on the top and a dry-etched microwell plate at the bottom. The microwells were coated with Pt(ll) meso-tetra(pentafluorophenyl)porphine (PtTFPP), an O2 sensing optode material and an O2-independent reference dye. A custom three dimensional (3D) printed holder facilitated the controlled horizontal movement (‘slipping’) of the channel plate over the microwell plate, thereby establishing or disrupting the fluid path over microwells. Collectively, these design elements enabled the immobilization of cells in microwells, their exposure to controlled fluid flows, the coordinated opening and closing of microwells and repeated measurements of single-cell O2 respiration. Uniquely, by sequentially executing opening and closing it became possible to measure single-cell respiration prior to and after exposure to chemical solutes. In a proof-of-concept application, we utilized SlipO2Chip™ to measure the impact of increasing exposures of the marine bacterial signal 2-heptyl-4-quinolone (HHQ) on the dark respiration of the diatom Ditylum brightwellii at single-cell resolution. Results revealed a dosedependent decrease in per-cell O2 dark respiration, with a maximum reduction of 40.2% observed at HHQ concentrations exceeding 35.5 M, and a half-maximal effective concentration (ECso) of 5.8 pM, consistent with that obtained via conventional bulk respiration methods. The ability of SlipO2Chip™ to sequentially assess the effects of chemical substances on single-cell O2 metabolism is advantageous for research where sample volumes are limited, such as clinical biopsies, studies involving rare microbial isolates, and toxicological studies wanting to address exposure effects while accounting for cell-to-cell variability.

[0134] Materials & Methods

[0135] Cell culture

[0136] Cultures of the diatom Ditylum brightwellii were purchased from the Bigelow culture collection (CCMP 3370). The size of D. brightwellii, ranged between 50 - 120 pm in length and 10 - 40 pm in width, as determined by a standard brightfield microscope. Cells were cultivated in L1 media and grown at a temperature of 22 °C while being subjected to a 14 / 10 h light / dark cycle. L1 media was prepared by dissolving 35 g of sea salt (Instant Ocean®, US) in 1 L of deionized (DI) water and by adding 1 mL of nitrogen (N), phosphorus (P), silicon (Si), trace metals stock solutions, respectively, and 0.5 mL of vitamin stock solution. All stock solutions were supplied in the L1 media kit (NCMA at Bigelow Laboratory, US). To ensure cultures were in mid-exponential growth for experiments, diatom cells were inoculated into fresh L1 media at a 1 :6 (volume / volume) dilution five days prior to experiments.

[0137] Device components and operations

[0138] SlipO2Chip™ is comprised of three main components: a bottom microwell plate decorated with O2- sensitive optodes for detecting respiration, a top channel plate for introducing fluids and a holder to aid in the assembly and slipping of the channel plate against the microwell plate. Details on parts of the 3D printed holder and assembly of SlipO2Chip™ are provided in Figs. 1A, 1 B, 2. Before assembling the microwell plate, polydimethylsiloxane (PDMS, 0.25 mm thickness, SuperClear silicone sheet, BISCO) and polyethylene terephthalate (PET, 0.05 mm thickness, Silflu 1 R88001, Siliconature S.P.A., US) films were precisely cut to match the shape and size of the channel plate using a desktop vinyl cutter (CAMM- 1 GS-24, Roland DGA Co., US). The elastic PDMS film was first affixed to the channel plate to enhance liquid-tightness. Subsequently, the gas-impermeable PET film was layered on top of the PDMS to ensure gas-tightness and facilitate smooth sliding. This combined PDMS-PET layering not only promoted seamless operation but also effectively sealed each microwell, minimizing cross-talk between them, as demonstrated in the sealing test (Figs. 1 C, 3). The microwell plate was secured in place by sandwiching it between two dedicated holder parts labeled ‘holder top’ and ‘holder bottom’ (Fig. 2). Following the secure attachment of the microwell plate into the holder, the channel plate + PDMS-PET film was carefully placed onto the microwell plate and affixed using six pairs of neodymium magnets, two pairs positioned in the middle and four pairs positioned at the corners of the top channel plate (Block magnet 10 x 3 x 2 mm and 20 x 4 x 3 mm, Supermagnete DE). The precise slipping movements, used for opening and closing microwells at the microscale, were facilitated by a 3D-printed holder and two commercial micrometer screws (High precision micrometer head, Thorlabs Inc., US), each offering a resolution of ± 1 m.

[0139] G / ass etching

[0140] The bottom microwell plate was built from a borosilicate glass wafer (Borosilicate 4-inch, thickness = 1000 ± 30 pm, MicroChemicals GmbH, DE). The wafer was first subjected to plasma cleaning and subsequently 7 pm of aluminium (Al) was deposited via a sputtering process (CS 730 S, Von Ardenne GmbH, DE) to serve as a future etch mask. After sputtering, the wafer was oven-dried at 200 °C for 5 min to eliminate residual moisture and enhance the adhesion of the photoresist. To fabricate the metal mask, a 5 pm layer of photoresist AZ-10XT (AZ® 10XT, Merck KGaA, DE) was spin-coated onto the Al layer. The photoresist was soft baked at 110 °C for 2 min and subjected to four cycles of UV light (365 nm) for 30 s each, utilizing a mask aligner (MA6 / BA6, Suss MicroTec GmbH, DE). Following UV exposure, the photoresist layer was developed (AZ 400K developer, Merck KGaA, DE) for 4 min and subsequently hard baked at 120 °C for 7 min. The Al layer was then etched using inductively coupled plasma - reactive ion etching (ICP-RIE) (SLR, PlasmaTherm LLC, US), yielding an Al mask comprising the desired microwell patterns. The AZ-10XT photoresist was removed using acetone (Acetone, VWR chemicals, US) and isopropanol (IPA) (2-propanol, VWR chemicals, US). The glass wafer, now patterned with the Al mask, was then processed in a reactive ion etching (RIE) machine (110 S / DE, Tegal Co., US) to etch the microwells to a depth of 50 pm. Following this process, the Al mask was removed using an Al etchant at 40°C, prepared from phosphoric acid (H3PO4 85%, Sunchem AB, SE), acetic acid (CH3COOH 99%, Sunchem AB, SE) and nitric acid (HNO3 69% VLSI selectipur, BASF, DE) in a volumetric ratio of 29 : 5 : 1 . To finalize the process, the glass wafer was immersed in buffered oxide etch (BHF, 7-1 , VWR, US) for 30 min to smooth the dry-etched surface, followed by thorough rinsing in deionized water. The entire dry etching protocol is depicted in Fig. 4A.

[0141] The top channel plate was fabricated from a fused silica glass wafer (Fused silica JGS2 wafer 4-inch, thickness = 350 ± 25 pm, MicroChemicals GmbH, DE), via a wet etching process. Initial preparation steps included plasma cleaning of the glass wafer, followed by the deposition of a 1 pm thick molybdenum (Mo) layer using a sputter (CS 730 S, Von Ardenne GmbH, DE) to act as an etch mask. After this deposition, the wafer underwent a hydrophobic treatment within a vapor prime furnace (Star 2000, Imtec Inc., US), followed by the application of a 1 pm thick layer of photoresist (MicropositTM S1813TM G2 photoresist, Dow Inc., US) via spin-coating. The wafer was then soft baked at 110 °C for 2 min and subjected to UV exposure for 6 s in the mask aligner. The exposed photoresist was developed for 45 s (MicropositTM 351 developer, Dow Inc., US) and hard baked at 120 °C for 5 min. The channel pattern was then transferred onto the Mo layer through etching in a prepared Mo etchant, comprising water, HNO3, and hydrochloric acid (HCI 36%, BASF, DE) mixed in a volumetric ratio of 30 : 57 : 13, for 3 min. To protect the backside of the wafer from this etching process, this side of the wafer was taped with acid- resistant tape (SPV224R PVC Surface Protection tape, Nitto Denko Co., JP). The channels were then etched into the glass substrate using 50% hydrofluoric acid (HF 50%, VWR, US) at an etch rate of 1 .5 pm min1for 37 min. After etching, the photoresist was removed through consecutive acetone and IPA baths, followed by a Mo etchant bath to eliminate the metal mask. The entire wet etching protocol is available in Fig. 4B.

[0142] After the etching process, wafer samples were sectioned using a dicing saw (DAD 361 , Disco Co., JP). Furthermore, 2 mm diameter access holes were introduced on the bottom microwell plate by a laser cutter (AIO G+ 532 nm 5 W, Ostling Marksystem AB, SE). The dimension and surface topography of etched structures were evaluated using a profilometer (Dektak 150 Stylus profiler, Bruker Nano Inc., US). Surface roughness was quantified in terms of the arithmetical mean roughness value (Ra), defined as the average of deviations from the mean line across the assessment length.

[0143] Optodes decoration and calibration

[0144] To functionalize the dry-etched microwells, the 02-sensitive optodes were deposited at the bottom of each microwell utilizing a film applicator tool. This procedure started with the preparation of an optode + reference dye solution with the following proportions: 1.5 mg PtTFPP (Frontier Scientific Inc., US), 1.5 mg MY yellow dye (Macrolex Fluorescent Yellow 10GN, Lanxess GmbH, DE), and 1 g of polystyrene (Sigma-Aldrich Inc., US) in 10 mL toluene (Sigma-Aldrich Inc., US) and 2 g of silicone rubber (ELASTOSIL® E4, Wacker Chemie AG, DE). Subsequently, 80 pL of this mixture was pipetted onto the microwell plate and spread evenly across via a 50 pm applicator (4-sided Bar 60, 50-200 pm, BYK- Gardner Gmbh, DE, Fig. 5). By evaporating the toluene solvent this resulted in the formation of an optode sensor film that adhered to the entire microwell plate. Excess sensor film outside the microwells was carefully removed using a scalpel. This ensured that only microwells were decorated with the optode composition. Notably, due to edge effects the majority of the optode material was immobilized onto the sides of microwells. This ‘ring’ of optode composition was 3 ± 1 pm thick. The optode material in the central part of the microwell was measured to be 1 ± 1 pm thick. The thickness was characterized by a profilometer (Dektak 150 Stylus profiler, Bruker Nano Inc., US).

[0145] Calibration entailed exciting optode-coated microwells with UV light (395 / 25 nm) for a duration of 50 ms across various O2 concentrations. Subsequently, the resulting optode-luminescence was captured using a red, green, blue (RGB) camera (DFK 33UX264 USB 3.0 color industrial camera, Image Source, DE). This setup was integrated into a fully automated inverted Nikon Ti2-E microscope equipped with a perfect focus system (Nikon Inc., JPN) and a UV light source (SpectraX Light Engine, Lumencor Inc., US).

[0146] To process the obtained RGB images, the pixel-wise ratiometric signals (r) of the red emission (attributable to the 02-sensitive optodes) and the green emission (emanating from the 02-insensitive reference MY dye) at different O2 concentrations were extracted via Fiji (Fiji image processing, v2.0 Schindelin et al., Nature Methods, 2019 9(7): 676-682) and subsequently analyzed through the application of the Stern-Volmer relationship:

[0147] Here r = signifies the ratio of luminescence intensities ( / ) between the red and green channels in Igreen the presence of O2, being a function of O2 concentration [O2] (% air saturation); r0denotes ratio between red and green luminescence intensities in the absence of O2; ksvrepresents the Stern-Volmer constant. The Stern-Volmer constants derived from each calibration set were used to transform the experimentally obtained luminescence signals into O2 concentrations.

[0148] By conducting a 7-point calibration experiment in the gas phase (Fig. 6), we determined that the performance of O2 optodes obeyed a linear fit (Fig. 1 F). Therefore, we performed simpler 2-point calibrations in the liquid phase for most calibrations.

[0149] Validation of liquid-tightness and gas-tightness

[0150] To validate the liquid tightness of the SlipO2Chip™, fluorescein was introduced using a low-pressure pump system (OB1 MK3+ low-pressure pump, Elvesys Co, France) at 30 mbar pressure. This was carried out within the fully assembled device, with microwells left open. The pressure was lowered to 10 mbar stepwise over the course of 1 min and the top channel plate was subsequently slipped horizontally to close microwells. The ability of SlipO2Chip™ to effectively isolate liquid flow was confirmed by observing minimal presence of fluorescein solution outside the microwells. All fluorescein imaging was conducted using an RGB camera under blue light excitation (440 / 20 nm) and fluorescein isothiocyanate (FITC) specific emission (525 nm).

[0151] To validate the gas-tightness of SlipO2Chip™, the bottom microwell plate was positioned inside a customized chamber designed for precise control over gas composition. The plate was partitioned such that half was covered by a PDMS film (0.25 mm thickness) and the other half by a PDMS-PET film (0.25 + 0.05 mm thickness), with the PET side facing the microwells. Initially, the microwell plate was exposed to compressed air (CA) for a duration of 10 min. Subsequently, 100% nitrogen gas (N2) was introduced to the chamber. During this stepwise gas exposure, O2 concentrations within the microwells were monitored every 2 min using the automated microscopy equipped with an RGB camera, documenting the changes over a 60-min period to assess the effectiveness of the films in maintaining gas integrity.

[0152] Repeated single-cell O2 respiration experiments

[0153] Experiments began with cell loading, followed by iterative opening and closing of the SlipO2Chip™ to measure respiration, with and without chemical solutes. For cell loading, a 100 pL aliquot of 1 :2 diluted mid-exponentially growing culture was manually pipetted into SlipO2Chip™ while microwells were open. Cells were then permitted to sediment into microwells. This procedure was repeated several times, in order to ensure that the occupancy of microwells exceeded 20%.

[0154] To facilitate the introduction of L1 media into SlipO2Chip™, the device inlet was connected to the low- pressure pump system. First, a pressure of 30 mbar was set to remove cells outside of microwells and subsequently the pressure was manually reduced over 1 min to a final pressure of 10 mbar. This procedure helped retain cells within microwells while effectively washing away excess cells outside of the microwells. Once a stable fluid flow was established, the system underwent a 10 min equilibration period. Subsequently, the channel plate was slipped horizontally to close microwells. To quantify the O2 respiration of cells isolated within microwells, all external light sources were deactivated. Subsequently, the microwells were exposed to a 50 ms long pulse of UV light (395 / 25 nm, power 35%) every 2 min over a duration of 16 min. The O2-dependent luminescence from the optode material was recorded using an RGB camera as described before. In each experiment, we utilized the ‘Large Image Scanning Mode’ within Nikon Imaging Software (NIS) Elements (AR 5.10.00, Nikon Inc., JPN) to image a total of 275 microwells. This approach generated RGB time-lapse sequences that captured changes in O2 signals among individual microwells. The O2 concentration within each microwell was calculated from the obtained ratiometric values r via the Stern-Volmer equation, utilizing the ksvconstant obtained from the prior calibration step. Finally, single-cell O2 respiration rates were calculated using a linear fit to the O2 consumption over the entire 16-min observation period.

[0155] After the first O2 respiration measurement, microwells were re-opened by slipping the channel plate and by introducing fully oxygenated L1 media into the channel. Cells in microwells were left for 10 min under continuous fluid flow (10 mbar pump pressure). Following this incubation, microwells were closed again to conduct a second O2 respiration measurement on the exact same cells. This step was repeated for a third time. In combination, this experimental protocol assessed the effect of three repeated slipping motions on single-cell O2 respiration rates.

[0156] Single-cell O2 respiration under HHQ exposure

[0157] To explore the impact of HHQ on the O2 respiration of diatoms, microwells were filled with individual cells following previously described protocols. Following cell loading, the microwells containing cells were sealed and baseline respiration rates were obtained. These baseline respiration rates were obtained under standard L1 media supplemented with dimethyl sulfoxide (DMSO) as a carrier control. Following these baseline measurements, the microwells were opened and the media in the channel replaced with L1 media augmented with a specific concentration of HHQ (0, 5, 10, 20 or 100 pM) and left to incubate for 10 min before closing microwells again. This process was iterated in five separate experiments to acquire per-cell respiration rates first under L1 media + carrier control, and subsequently under L1 media + different concentrations of HHQ. Switching between L1 media + DMSO and L1 media + HHQ was achieved through a manual three-way switching valve located upstream of the channel inlet. Experimental data, both before and after exposure to the different HHQ concentrations, was used in a dose-response model and used to determine EC50 values via the following relationships: p _ p 1 _ rimax—Emin_ c —cmin "T1+ 10(iogECso-[dose])p where E denotes the reduction of O2 respiration rates for single-cells after HHQ exposure, i.e., 1 -

[0158] - - . Eminrepresents the minimal reduction in O2 respiration after HHQ O2respiration ratebefore doseexposure, while Emaxrepresents the maximum reduction in O2 respiration after HHQ exposure. The term [dose] refers to the concentration of administered HHQ. EC50 is identified as the half-maximal effective concentration of HHQ dose producing 50% of the maximal effect. The p value is the hill slope and quantifies the steepness of the dos-response curve. Bulk O2 respiration measurements

[0159] To benchmark the precision and accuracy of single-cell respiration measurements under HHQ exposures, the O2 respiration of the same diatom species was measured in commercial O2 sensing vials (OXVIAL4, PyroScience GmbH, DE). For each experiment, this entailed preparing a pair of 15 mL Falcon tubes, each filled with 5 mL of cell suspension at a density of 85 - 350 cells mL1. To one of the tubes, HHQ stock solution was added to achieve final concentrations of 0, 2.5, 5, 10, or 50 pM, while an equivalent volume of DMSO was added to the other tube as a carrier control. Both tubes were gently shaken for 10 min, and their contents were carefully transferred into two O2 sensing vials, ensuring no air was trapped in the headspace. A small stirring bar was added to each vial and the vials were placed on a magnetic stirrer for constant mixing during the measurement. The O2 concentration within each vial was monitored using a fiber-optic cable connected to a commercial 4-channel O2 sensing system (FireSting, Pyroscience GmbH, DE). This facilitated measurements of O2 consumption for the entire diatom population. Subsequent to the experiments, the cell numbers in each vial were manually counted using a Neubauer Counting Chamber and the resulting numbers used to calculate the per-cell respiration rates.

[0160] Results

[0161] SlipO2Chip™ allowed for the precise quantification of O2 respiration rates in single-cells under the coordinated absence and presence of user-controlled chemical solutes. SlipO2Chip™ integrated a movable channel plate on the top and a fixed microwell plate at the bottom. Embedded within the channel plate were three wet-etched fluidic channels, each 3.5 cm in length, 8 mm in width, and 70 pm in depth (Figs. 1A and 1 B). These channels were strategically positioned to overlay a microwell array consisting of 25 x 155 microwells (total n = 3875) when moved horizontally into the ‘open’ position. The microwells had a diameter of 140 pm, a depth of 50 pm, and a pitch (center-to-center spacing) of 100 pm. The bottom of microwells had a roughness (Ra) of 200-1000 nm, and when coated with O2-sensitive optode composition, enabled for measuring single-cell respiration rates when fully closed by horizontally moving the top plate (Fig. 7A). By utilizing strong neodymium magnets and a PDMS-PET film, SlipO2Chip™ reliably isolated individual microwells from both fluid flow (Fig. 1 C) and atmospheric O2 (Fig. 3B). Fig. 7 illustrates the stepwise operation of SlipO2Chip™, showcasing how the horizontal motion of the channel plate enabled the opening and closing of microwells and how this facilitated cell loading, the introduction of chemical solutes, and measurements of single-cell respiration. Cell loading typically resulted in 20 - 60 % of microwells being occupied by cells, with 80 - 90 % of filled microwells containing single-cells, 10 - 20 % containing doublets, while triplet occupation was rarely observed. Opening and closing SlipO2Chip™ has negligible effects on single-cell respiration

[0162] To assess whether the process of mechanically opening and closing microwells would cause stress to cells within microwells we conducted a simple test. This involved subjecting individual diatom cells (Ditylum brighwellii, n = 18) to three consecutive opening and closing cycles while measuring their respiration rate three times (Fig. 8A). This test revealed that the second opening and closing cycle resulted in a significant increase in respiration rates (by 6.4 ± 3.7 %, p = 0.0052, Mann-Whitney test) compared to the first opening and closing cycle. Further, the third opening and closing cycle resulted in a significant reduction of respiration rates (by 33.4 ± 5.0 %, p < 0.00001) compared to the second cycle. The results of this straightforward experiment suggested that opening and closing microwells twice had minimal impact on O2 respiration rates in single diatom cells. However, conducting a third cycle led to notably significant changes in respiration rates. This effect could stem from mechanical stress arising from repeated slipping motions, potentially damaging the fragile silica shell of diatoms, or a combination of factors such as nutrient depletion or waste accumulation in the microwells. For this study, we had found that two consecutive opening and closing cycles were not only sufficient but also minimally impacted diatom cells, effectively measuring the effects of chemical exposure both before and after treatment. Consequently, we had standardized this dual-cycle protocol for all subsequent experiments to ensure consistent and reliable data collection.

[0163] HHQ affects single-cell diatom respiration in a dose-dependent manner

[0164] To evaluate the efficacy of SlipO2Chip™ in orchestrating the exposure of single-cells to chemical solutes and measuring their impact on individual cell respiration, we designed an experiment using the bacterial infochemical HHQ. This molecule is a recognized quorum sensing signal produced by bacteria and plays a crucial role in their interactions with host phytoplankton. To assess the impact of HHQ on diatom respiration, we first exposed individual D. brightwellii cells within SlipO2Chip™ to L1 media + carrier control. These baseline measurements revealed respiration rates ranging between 3.2 x 109and 8.1 x 109pmol 02' cell1■ min1(Fig. 9A, ‘before dose’). While the range of these rates is comparable to respiration measurements conducted in bulk, both by us and by others on other diatom species (Table 1), they also underscore the significant cell-to-cell variability that is often overlooked in traditional bulk measurements. Table 1 - Single-cell dark respiration rates of different diatom species measured using bulk approaches After these baseline measurements, we introduced L1 media augmented with one of five concentrations of HHQ (0, 5, 10, 20 or 100 pM) into the main channel of SlipO2Chip™ and exposed cells for 10 min. Following this exposure period, microwells were closed and the respiration rates of each cell were measured again. We note that for each concentration of HHQ this involved a different set of immobilized diatom cells, since we can only execute two consecutive opening / closing cycles to ensure reliable results. Although different cells were used, the results of this experiment revealed a consistent decrease in respiration rates with increasing HHQ concentrations (Fig. 9A). Using single-cell measurements on baseline respiration (L1 media + carrier control) and post-exposure respiration (L1 + HHQ at varying concentrations), we calculated E values, which is the ratio of the reduction of respiration rate after chemical exposure and before exposure. E thus provides a straightforward measure of the inhibitory effect of HHQ on respiration of single diatoms by comparing it to its individual baseline respiration. Through this comparison, we observed a reduction in respiration rates of 2.7 ± 6.1 % (0 pM HHQ, n = 26), 17.3 ± 8.0 % (5 pM HHQ, n = 39), 34.7 ± 7.7 % (10 pM HHQ, n = 71), 38.0 ± 5.7 % (20 pM HHQ, n = 36), and 41.1 ± 5.6 % (100 pM HHQ, n = 57) following exposure to HHQ. In order to further explore the effect of HHQ on diatom respiration, we fitted a classical dose-response model on per-cell values of E (Fig. 9C, Table 2). This revealed a maximal reduction of single-cell respiration (Emax, singie-ceii) by 40.2 ± 1.4 % compared to cells not exposed to HHQ at a concentration of 35.5 pM. Using the same doseresponse relationship, we also determined the half-maximal effective concentration (ECso, single-cell) for single-cells to be 5.8 ± 0.5 pM. These ECso values, obtained from separate populations of individual cells before and after HHQ exposure, closely align with previously reported ECso values for HHQ in diatom species such as C. closterium, P. tricornutum, and A. minutissimum, ranging from 1 .4 pM to 4.9 pM (Dow et al., ChemBioChem, 2020, 21 : 1206-1216).

[0165] Table 2 - Dose-response parameters derived from single-cell data and bulk measurements

[0166] To further validate the accuracy of our single-cell dose-response relationship, we conducted additional measurements of D. brightwelliii under increasing HHQ concentrations using standard commercial respiration vials. This revealed that, in bulk, respiration is maximally reduced by approximately 34.2 ± 1 .3 % (Emax, bulk) at an HHQ concentration of 28.2 pM, compared to the control. Additionally, we determined the half-maximal effective concentration in bulk to be 4.1 ± 0.4 pM, which closely aligns with the earlier ECso values of 5.8 ± 0.5 pM obtained for single-cells. Collectively, these findings demonstrate that HHQ has a stronger inhibitory effect on diatom respiration in single-cell measurements, with an Emax, single-cell Of 40.2 ± 1 .4 %, compared to bulk measurements, which show an Emax, bulk of 34.2 ± 1 .3 %. This discrepancy may stem from undetected cell-to-cell variabilities in bulk measurements, potentially leading to the emergence of chemically tolerant subpopulations that are only discernible through single-cell analyses. The findings underscore the significance of single-cell techniques, like SlipO2Chip™, in evaluating these effects with a level of resolution typically absent in (eco)toxicological investigations. Additionally, singlecell measurements conducted also revealed a higher response efficiency (psingie-ceii = 2.9 ± 0.8 vs. pb ik = 2.7 ± 0.6), indicating a greater effectiveness of HHQ at the single-cell level. This effect could stem from the absence of cell-to-cell interactions and competition for resources within microwells, characterized by uniform exposure to surrounding media and limited chemical interchange between neighboring microwells. This stands in contrast to bulk environments, where cells encounter each other stochastically through mixing and collectively deplete nutrient levels. While bulk measurements, thus, offer rapid insights into collective effects of chemical exposure, single-cell analyses provide greater precision in measuring individual cell responses to chemical challenges.

[0167] EXAMPLE 2

[0168] To assess the compatibility of fluorinated oil coating with SlipO2Chip™, optode-coated microwells were first filled with water containing a green-fluorescent dye (fluorescein) to facilitate visualization (Fig. 17A). Following aqueous loading, a layer of FC-40 was dispensed onto the top surface of the microwell array followed by scraping to remove the excess.

[0169] Upon the addition of FC-40, substantial disruption of the aqueous phase within the microwells was observed, as evidenced by displacement of the initially filled green dye solution (Fig. 17B). This is likely due to the interfacial tension between the fluorinated oil (FC-40), reagent in the well and the optodecoated microwells, and led to a notable reduction in experimental yield. This data indicates that the application of a FC-40 coating between the first main surface 12 of the microwell plate 10 and the first main surface 34 of the channel plate 30 in SlipO2Chip™ led to crosstalk between the microwell 20. Accordingly, a FC-40 coating will not isolate the microwells 20 but rather induced contamination between adjacent microwells 20 in the microwell plate 10.

[0170] EXAMPLE 3

[0171] To assess the gas permeability of FEP as a sliding polymer film or coating for the SlipO2Chip™, optodecoated microwells were filled with air-saturated water and sealed with a FEP film. The chip was then exposed to 100% N2gas, and the oxygen concentration within the microwells was monitored every 2 minutes using automated microscopy and an RGB camera. The resulting oxygen depletion profile was compared to previously reported data using PDMS and PDMS+PET lids (see Example 1 and Fig. 3B), where 100% N2was introduced at 10 minutes. The results are presented Fig. 18, which shows that PDMS-PET is gas-impermeable, whereas FEP is not gas-impermeable.

[0172] The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible.

Claims

CLAIMS1. A monitoring device (1) comprising: a microwell plate (10) comprising a plurality of microwells (20) dimensioned to hold a respective biological entity and arranged in a first main surface (12) of the microwell plate (10); a channel plate (30) comprising at least one fluid channel (40, 42) arranged in a first main surface (34) of the channel plate (30) and in fluid connection with a fluid input port (11 ; 11 A, 11 B) and a fluid output port (13; 13A, 13B) in a loading position; and an optode composition (25) present in the microwells (20) and comprising an analyte-sensitive optode compound generating a detectable optical signal in response to an analyte, wherein the channel plate (30) and the microwell plate (10) are slidably arranged together and movable relative to each other between the loading position, in which the at least one fluid channel (40, 42) is in fluid connection with at least a subset (21, 23) of the plurality of microwells (25), and a monitoring position, in which the channel plate (30) closes the at least a subset (21 , 23) of the plurality of microwells (20); and at least the portion of the microwell plate (10) comprising the plurality of microwells (20) is optically transparent and / or at least the portion of the channel plate (30) aligned with the at least a subset (21 , 23) of the plurality of microwells (20) in the monitoring position is optically transparent.

2. The monitoring device according to claim 1 , wherein the channel plate (30) comprises multiple fluid channels (40, 42) arranged in the first main surface (34) of the channel plate (30); each fluid channel (40, 42) of the multiple fluid channels (40, 42) is in fluid connection with a respective fluid input port (11 A, 11 B) or a common fluid input port (11 ) and a respective fluid output port (13A, 13B) or a common fluid output port (13) in the loading position; and each fluid channel (40, 42) of the multiple fluid channels (40, 42) is in fluid connection with a respective subset (21, 23) of the plurality of microwells (20) in the loading position.

3. The monitoring device according to claim 1 or 2, wherein the microwell plate (10) comprises at least one matrix (21, 23) of microwells (20) arranged in the first main surface (12) of the microwell plate (10).

4. The monitoring device according to any one of claims 1 to 3, further comprising a gas-impermeable sliding polymer film or coating (50) attached to the first main surface (34) of the channel plate (30), wherein the gas-impermeable sliding polymer film or coating (50) is in slidable contact with the first main surface (12) of the microwell plate (10).

5. The monitoring device according to claim 4, wherein the gas-impermeable sliding polymer film or coating (50) is fluid tight and forms a fluid tight seal of individual microwells (20) in the at least a subset (21, 23) of the plurality of microwells (20) in the monitoring position.

6. The monitoring device according to claim 4 or 5, wherein the gas-impermeable sliding polymer film or coating (50) comprises: a liquid-tight film or coating of a first polymer, preferably polydimethylsiloxane, attached to the first main surface (34) of the channel plate (30); and a gas-impermeable film or coating of second polymer, preferably polyethylene terephthalate, attached to the liquid-tight film or coating.

7. The monitoring device according to any one of claims 1 to 6, wherein the optode composition (25) is deposited on at least a portion of the surfaces (22, 24) of the microwells (20).

8. The monitoring device according to any one of claims 1 to 7, wherein the optode composition (25) also comprises an analyte-insensitive reference compound, preferably 3-(5-chlorobenzoxazol-2-yl)-7- diethylaminocoumarin, capable of generating a detectable optical reference signal.

9. The monitoring device according to any one of claims 1 to 8, wherein the optode composition (25) also comprises polystyrene and preferably silicone rubber.

10. The monitoring device according to any one of claims 1 to 9, wherein the analyte-sensitive optode compound is selected from the group consisting of an oxygen-sensitive optode compound, a pH-sensitive optode compound, and a carbon dioxide-sensitive optode compound, preferably selected from the group consisting of Pt(ll) meso-tetra(pentafluoropheynyl)porphine, a BF2-chelated tetraarylazadipyrromethene, and 4,4'-(5, 5-difl uoro-1 , 9-dip henyl-5H-4X4, 5X4-dipyrrolo-[1 , 2-c: 2', 1 '- / ] [1 , 3, 5, 2]triazabori ni ne-3, 7- diyl)diphenol.11 . The monitoring device according to any one of claims 1 to 10, wherein the microwell plate (10) is made of an optically transparent material, preferably glass, and more preferably borosilicate glass.

12. The monitoring device according to any one of claims 1 to 11 , wherein the channel plate (30) is made of an optically transparent material, preferably glass, and more preferably fused silica glass.

13. The monitoring device according to any one of claims 1 to 12, wherein the microwell plate (10) comprises the fluid input port (11 ; 11 A, 11 B) in a first end surface (15) and the fluid output port (13; 13A, 13B) in a second, opposite end surface (17); the fluid input port (11; 11 A, 11 B) is in fluid connection with a first opening (16; 16A 16B) in the first main surface (12) of the microwell plate (10); the fluid output port (13; 13A, 13B) is in fluid connection with a second opening (18; 18A, 18B) in the first main surface (12) of the microwell plate (10); and the at least one fluid channel (40, 42) is in fluid connection with the first opening (16; 16A, 16B) and the second opening (18; 18A, 18B) in the loading position.

14. A monitoring system (100) comprising: a monitoring device (1) according to any one of claims 1 to 13; a light system (110) comprising a light source (112) and configured to direct input light (111 ) into the at least a subset (21, 23) of the plurality of microwells (20); and a detector (120) configured to detect output light (113, 115) emitted by the optode composition (25) in response to the input light (111) and based on the analyte.

15. A method of monitoring biological entities comprising:(a) loading biological entities into microwells (20) of the monitoring device (1) according to any one of claims 1 to 13;(b) moving the channel plate (30) relative to the microwell plate (10), or vice versa, to the monitoring position;(c) directing input light (111) into the at least a subset (21, 23) of the plurality of microwells (20);(d) detecting, for each microwell (20) of the at least a subset (21, 23) of the plurality of microwells (20) comprising at least one biological entity, output light (113, 115) from the microwell (20);(e) generating, for each microwell (20) of the at least a subset (21 , 23) of the plurality of microwells (20) comprising at least one biological entity, a reference value based on the output light (113, 115) detected in (d);(f) moving the channel plate (30) relative to the microwell plate (10), or vice versa, to the loading position;(g) flowing a fluid through the at least one fluid channel (40, 42) in the loading position;(h) moving the channel plate (30) relative to the microwell plate (10), or vice versa, to the monitoring position;(i) directing input light (111) into the at least a subset (21 , 23) of the plurality of microwells (20);0) detecting, for each microwell (20) of the at least a subset (21 , 23) of the plurality of microwells (20) comprising at least one biological entity, output light (113, 115) from the microwell (20);(k) generating, for each microwell (20) of the at least a subset (21 , 23) of the plurality of microwells (20) comprising at least one biological entity, a measurement value based on the output light (113, 115) detected in 0); and(l) determining an analyte response of biological entities to the fluid based on the measurement value and the reference value determined for each microwell (20) of the at least a subset (21, 23) of the plurality of microwells (20) comprising at least one biological entity.

16. The method according to claim 15, wherein (a) loading the biological entities comprises: adding a suspension of biological entities to microwells (20) of the monitoring device (1) according to any one of claims 1 to 13 in an open position, in which the channel plate (30) is not covering the at least a subset (21, 23) of the plurality of microwells (20) in the microwell plate (10); moving the channel plate (30) relative to the microwell plate (10), or vice versa, to the loading position; and flowing a culture medium through the at least one fluid channel (40, 42) in the loading position.

17. The method according to claim 15, wherein (a) loading the biological entities comprises: flowing a suspension of biological entities through the at least one fluid channel (40, 42) in the loading position; and flowing a culture medium through the at least one fluid channel (40, 42) in the loading position.

18. The method according to claim 16 or 17, wherein flowing the culture medium comprises flowing the culture medium through the at least one fluid channel (40, 42) in the loading position at a first fluid pressure and reducing the fluid pressure to a second fluid pressure that is lower than the first fluid pressure while continuing to flow the culture medium through the at least one fluid channel (40, 42).

19. The method according to any one of claims 15 to 18, wherein the optode composition (25) also comprises an analyte-insensitive reference compound capable of generating a detectable optical reference signal;(d) detecting the output light (113, 115) comprises, detecting, for each microwell (20) of the at least a subset (21 , 23) of the plurality of microwells (20) comprising at least one biological entity , output light(113) emitted by the analyte-sensitive optode compound and reference output light (115) emitted by the analyte-insensitive reference compound from the microwell (20);(e) generating the reference value comprises generating, for each microwell (20) of the at least a subset (21 , 23) of the plurality of microwells (20) comprising at least one biological entity , the reference value based on the output light (113) and the reference output light (115) detected in (d);0) detecting the output light (113, 115) comprises detecting, for each microwell (20) of the at least a subset (21 , 23) of the plurality of microwells (20) comprising at least one biological entity , output light (113) emitted by the analyte-sensitive optode compound and reference output light (115) emitted by the analyte-insensitive reference compound from the microwell (20); and(k) generating the measurement value comprises generating, for each microwell (20) of the at least a subset (21, 23) of the plurality of microwells (20) comprising at least one biological entity , the measurement value based on the output light (113) and the reference output light (115) detected in (j).

20. The method according to any one of claims 15 to 19, wherein (I) determining the analyte response comprises determining the analyte response of biological entities to the fluid based on a ratio between the measurement value and the reference value determined for each microwell (20) of the at least a subset (21, 23) of the plurality of microwells (20).

21. The method according to claim 20, wherein (I) determining the analyte response comprises determining a concentration of the analyte ] based on the equation:wherein r represents the measurement value, r0represents the reference value, and ksvrepresents the Stern-Volmer constant.

22. The method according to any one of claims 15 to 21 , further comprising: moving the channel plate (30) relative to the microwell plate (10), or vice versa, to an open position, in which the channel plate (30) is not covering the at least a subset (21 , 23) of the plurality of microwells (20) in the microwell plate (10); and retrieving biological entities from microwells (20) of the at least a subset (21 , 23) of the plurality of microwells (20).

23. A method for producing a monitoring device (1), the method comprising: generating, in a first substrate, a plurality of microwells (20) dimensioned to hold a respective biological entity to form a microwell plate (10) comprising the plurality of microwells (20) in a first main surface (14) of the microwell plate (10); generating, in a second substrate, at least one fluid channel (40, 42) to form a channel plate (30) comprising the at least one fluid channel (40, 42) in a first main surface (34) of the channel plate (30); depositing an optode composition (25) in the plurality of microwells (20), wherein the optode composition (25) comprises an analyte-sensitive optode compound generating a detectable optical signal in response to an analyte; and slidably arranging the channel plate (30) and the microwell plate (10) together with the first main surface (34) of the channel plate (30) facing the first main surface (12) of the microwell plate (10) so that the channel plate (30) and the microwell plate (10) are movable relative to each other between a loading position, in which the at least one fluid channel (40, 42) is in fluid connection with at least a subset (21, 23) of the plurality of microwells (20), and a monitoring position, in which the channel plate (30) closes the at least a subset (21 , 23) of the plurality of microwells (20), wherein at least the portion of the microwell plate (10) comprising the plurality of microwells (20) is optically transparent and / or at least the portion of the channel plate (30) aligned with the at least a subset (21 , 23) of the plurality of microwells (20) in the monitoring position is optically transparent.

24. The method according to claim 23, further comprising attaching a gas-impermeable sliding polymer film or coating (50) attached to the first main surface (34) of the channel plate (30), wherein the sliding polymer film or coating (50) is in slidable contact with the first main surface (12) of the microwell plate (10).

25. The method according to claim 24, wherein attaching the gas-impermeable sliding polymer film or coating (50) comprises: attaching a liquid-tight film or coating of a first polymer, preferably polydimethylsiloxane, to the first main surface (34) of the channel plate (30); and attaching a gas-impermeable film or coating of second polymer, preferably polyethylene terephthalate, to the liquid-tight film or coating.

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