Incubator integrated electrochemical analysis platform

Abstract

The invention relates to an electrochemical analysis method and platform compatible with screen-printed electrodes (SPE) (27) and any potentiostat (2) designed for cell-based tests, enabling cells to maintain healthy proliferation while being shielded from external influences.

Description

[0001] INCUBATOR INTEGRATED ELECTROCHEMICAL ANALYSIS PLATFORM

[0002] Technical Field

[0003] The invention relates to an incubator integrated electrochemical analysis platform.

[0004] The invention particularly relates to an incubator system developed to prevent cell-based studies conducted with screen printed electrodes (SPE) from being affected by the external environment.

[0005] Known State of the Art

[0006] Electrochemical analysis methods have become one of the most preferred analysis methods in recent years due to their advantages such as fast response time, high sensitivity in analyte detection, and ability to work with low-cost devices (Wang, 2006). Electrochemical analysis methods generally consist of an electrolytic solution containing the substance to be analyzed; a three-electrode configuration consisting of a working electrode, a reference electrode and a counter electrode; and a transducer mechanism connecting this electrode system to each other. The potential of working electrodes changes depending on the concentration of the analyte of interest and possesses the ability to be polarized. The most commonly used working electrodes are platinum, gold and carbon electrodes. The counter electrode allows current to pass through itself to transfer electricity from the solution to the working electrode and completes the 3-electrode configuration. Ag / Ag+, Ag / AgCI and saturated calomel electrodes are widely used as reference electrodes.

[0007] In recent years; Screen Printed Electrodes (SPEs), which integrate working, reference and counter electrodes in a compact substrate, have emerged as a result of developments in screen printing technology (Hayat et al. 2013). SPEs are highly preferred in electrochemical measurements due to their easy-of-use and suitability for miniaturization. SPEs are disposable, small, portable, compact and ergonomic electrodes, offering a combined structure that makes them suitable for a wide range of applications including analytical chemistry, drug control, clinical and environmental analysis. The integration of screen printing technology into electrochemical analysis methods has led to the development of combined systems, successfully commercialized into cost-effective and rapid analytical devices. As a result of all these developments, electrochemical analysis methods have become frequently preferred in different areas, e.g., environmental and food analysis, medical diagnosis and detection, waste control, cellular analysis and agriculture (Ferrari et al. 2021).

[0008] Cyclic Voltammetry (CV), Differential Pulse Voltammetry (DPV), Square Wave Voltammetry (SWV), which involve electron-mediated processes based on the measurement of electrical signals such as current, resistance, or potential during direct electron transfer, along with Electrochemical Impedance Spectroscopy (EIS), which relies on the assessment of electron transfer resistance, are among the most commonly utilized techniques in electrochemical measurement systems.

[0009] In recent years, electrochemical analysis methods have garnered significant attention in cell-based studies due to their advantages. Particularly, in research focused on understanding the mechanisms of living systems, in-vitro cell-based models that mimic the environmental conditions of biological systems have been developed (Van Duinen et al. 2015). In another study, it has been reported that cell proliferation closely correlates with impedance corresponding to the numbers of cell adhered on the SPE surface (Arias et al. 2010), and that these values can also be quantified using potentiometric techniques integrated with impedance measurements (Wu et al. 2013; Xu et al. 2016). It is widely recognized that the impedance increases proportionally with the number of viable cells; while the loss of membrane integrity during cell death results in a decrease in impedance (Yalgin et al. 2021). Following the development of the cell-substrate impedance sensing technique by Tran et al. to monitor adherent cells, various studies have utilized with the Cell-Substrate Impedance Sensing (ECIS) technique to determine real-time cell viability for drug screening (Tran et al. 2013).

[0010] Many studies have reported that the cell viability on the electrode surface is maintained for a certain period of time (Zhang et al. 2017; Arias et al. 2010; Su et al. 2014; Sun et al. 2018). In a similar study, it was reported that cell viability was preserved after the application of specific potentials to the electrode, enabled by an apparatus placed on the SPE, which also effectively prevented external factors from affecting the cells (Guette-Marquet et al. 2022).

[0011] In another study, Winkler et al. reported that 3-dimensional printed components (to produce the apparatus to integrate with SPEs) have no negative effects on cell types (Winkler et al. 2022), while designs utilizing Polydimethylsiloxane (PDMS) enabled label-free, real-time monitoring of cell monolayers using electrical cell-substrate impedance, with cell viability maintained throughout the monitoring period (Zhou et al. 2021; Pan et al. 2019; We et al. 2022).

[0012] Despite the wide range of applications of cell-based electrochemical analyses; in electrochemicalbased studies, cells are examined under laboratory conditions different from the incubation conditions. The differences in the test environment compared to the incubator environment can affect the clustering of cells (Ma and Lim 2003), and cause oxidative damage in the cells. These variations could affect the uncertainty of the electrochemical behavior of cells and prevent the correct interpretation of the data. In addition, cells taken from the cell culture environment (e.g., 37°C and 5% CO2) can be stressed due to the low temperature and CO2levels, (e.g., between 16 and 20°C; and 0.5% C02). The changes in cell behavior or proliferation caused by the external factors described here reduce the accuracy and reliability of cell-based tests.

[0013] Electrochemical techniques have a variety of applications including blood glucose analysis, environmental analysis, medical diagnosis, waste control, food analysis, agriculture and molecular analysis. Recently, electrochemical techniques developed based on the methods used in these applications have also begun to be utilized frequently in cellular analyses. Although many significant scientific data and original insights into cellular behaviors and intracellular pathways have been derived from these studies, there are some critical issues related to the platforms used in the analyses. In these studies; conducting cellular analyses in a laboratory environment where incubation conditions are not maintained leads to the exposure of cells to external factors thereby reducing the accuracy of the cellular data obtained from the tests.

[0014] The patent application US2018291417A1 relates to a microprocessor-controlled microfluidic platform technology including cell incubators, microarrays, microfluidic systems, as well as miniaturized biochemical and chemical detectors. The key distinction between the invention described in the patent application "Incubator integrated electrochemical analysis platform" and the technology covered byUS2018291417Al lies in the methods and the measurement platform employed for analyzing cellular behaviors. In the same patent application, the behavior of cells is monitored in a microplate using an optical-based imaging system while the CO2, humidity and temperature parameters are maintained in the cell incubator.

[0015] The patent CN100367033C describes a microfluidic chip-integrated device with a high-efficiency constant temperature system. This technology integrates a constant temperature device directly into a capillary electrophoresis chip system, benefiting from the high-efficiency constant temperature provided by the thermostat. The invention subject to the present patent application is designed to perform electrochemical analyses of biological systems while maintaining cells under optimal conditions. In contrast, the invention described in patent CN100367033C focuses on providing temperature control for microfluidic chips used in chemical reactions or biochemical analyses.

[0016] The patent application number CN111812167A relates to the field of electrochemical sensing and microfluidic control, specifically focusing on a platform and its application for chemical indirect toxicity detection. The objective of this patent application is to integrate electrochemical biosensor analysis technology with microfluidics to enable high-throughput analyses, and to incorporate various microarray analysis techniques based on microfluidic technology.

[0017] The patent application number CN111999362A describes an electrochemical sensor capable of analyzing the toxicity of heavy metal ions via a smartphone, with the research study focused on food- quality analysis and detection. This invention does not incorporate a protective device, corrosionresistant material or anti-corrosion coating to protect and prevent the corrosion of the connector ends of the SPEs. As a result, the lack of protective measures affects the performance of the SPE, increasing the risk of false or unreliable signals.

[0018] In the existing literature on cell-based analyses conducted with the current electrochemical techniques, there is no system that is both integrated with an incubator and capable of maintaining cells on the electrode surface, while simultaneously enabling the monitoring of live cells on the electrode surface using electrochemical techniques.

[0019] Brief Description and Purpose of the Invention

[0020] The invention described in the patent application discloses an electrochemical analysis platform where SPE-based analyses are conducted for cell-based tests. This platform is compatible with all types of potentiostats and enables the observation of cellular behaviors of living cells on the SPE surface using electrochemical methods. It maintains carbon dioxide (CO2) and temperature parameters within the incubator environment, simulating in-vivo conditions, and ensures the healthy proliferation of cells while protecting from exposure to external factors.

[0021] To address the limitations of existing technologies, the invention facilitates real-time electrochemical analysis of cells seeded on the electrode surface through an electrochemical analysis platform integrated with an incubator. The SPE within the incubator, where cells adhere to the surface, are supplied with cell and analysis solutions via a flow pump integrated into the system. This configuration ensures healthy cell proliferation during testing, thereby improving the reliability and accuracy of the tests. Another objective of the invention is to ensure that the temperature, carbon dioxide and sterilization conditions required for maintaining cell viability and proliferation are provided through the Incubator Module. In the Microfluidic Module; an elastic material-based flow box, where the electrochemical analyses of the cells are performed, seals the SPE surface with a sample holder that ensures the flow box remains securely fixed he necessary solutions and reagents are infused into the flow box via pump controlled by a microcontroller. The Electrochemical Test Module performs SPE- based electrochemical analyses, while the User Interface Module provides direct controls over the pump system, incubator electronics, and Electrochemical Test Module.

[0022] The invention aims to establish stable conditions for maintaining the parameters essential for the optimal state of the cells seeded on the SPE surface, thereby enabling the accurate extraction of cellular behaviors through electrochemical data. The User Interface Module of the invention controls the hardware components, while the integrated software processes the raw data obtained from the potentiostat of the Electrochemical Test Module, converting this data into meaningful information.

[0023] Another objective of the invention is to enhance the accuracy of the data obtained by performing electrochemical methods under incubation conditions, which minimizes the exposure of cells to external environmental factors through the incubator-integrated approach. Unlike the flow cells commonly used for SPE-based electrochemical analyses, the invention reduces oxidative damage caused by external environments and stress effects resulting from the sudden changes by incorporating a three-dimensional flow box into the incubator. The software of the User Interface Module facilitates user-friendly test processes.

[0024] The invention aims to maintain the viability and proliferation of live cells on the SPE surface within an incubator environment while enabling real-time examination of their behaviours using electrochemical methods. The invention incorporates a potentiostat device that facilitates cell-based electrochemical measurements with SPEs.

[0025] Another objective of the invention is to enable the integration of SPEs into an incubator casing, which distinguishes it from existing SPE-based technologies, thus allowing the electrochemical analysis of cellular behaviors within the incubator environment. In this way, while ensuring the viability and proliferation of cells, the invention prevents the effect of external factors on cellular behavior and consequently, on the electrochemical measurement results, ensuring the acquisition of highly accurate electrochemical data.

[0026] Additionally, the invention aims to protect the proliferation capacity of the cells by delivering fresh medium to the cells on the SPE surface via a microfluidic pump, in conjunction with the incubator included in the technology. Furthermore, the invention allows drug-containing media to the cellular environment through the pump system without external intervention, enabling the investigation of drug effects on cell proliferation.

[0027] Another objective of the invention is to control the potentiostat device, microfluidic pump and incubator in the system via a single user interface, allowing all protocols to be fully automated with a single software. This feature minimizes user errors in multi-step and complex electrochemical analyses.

[0028] The invention is compatible with all commercially available potentiostats. Upon integrating a commercial potentiostat into the system, the system software automatically detectss the new potentiostat and generates raw data through its operation. Additionally, the software actively controls the incubator and the heating box which maintains solutions at optimum parameters. During the measurements, the pump-valve system, which continuously supplies fresh medium to the cell environment, is automatically activated along with the system. The operator prepares the cells seeded on the SPE surface for electrochemical analysis by incubating them in a commercial incubator using the sample preparation apparatus, an integral component of the invention. Finally, the SPE, placed in the flow box, is integrated into the incubator casing, and the raw data obtained through the software is processed and transformed into meaningful information.

[0029] Description of Figures

[0030] Figure 1: General operational view of the incubator-integrated electrochemical analysis platform described in the invention.

[0031] Figure 2: General view of the flow box of the incubator-integrated electrochemical analysis platform described in the invention.

[0032] Figure 3: General view of the sample preparation apparatus of the incubator-integrated electrochemical analysis platform described in the invention.

[0033] Explanation of Reference Numbers

[0034] 1. Microcontroller [for pumps and valves]

[0035] 2. Potentiostat

[0036] 3. Microcontroller [for heating box]

[0037] 4. Microcontroller [for incubator casing]

[0038] 5. Heating box

[0039] 6. SPE connector

[0040] 7. Driver board [for valves]

[0041] 8. Valve

[0042] 9. Manifold

[0043] 10. CO2sensor

[0044] 11. Temperature sensor [for incubator casing]

[0045] 12. Driver board [for heating box]

[0046] 13. Driver board [for incubator casing]

[0047] 14. Temperature sensor [for heating box]

[0048] 15. Heating pad [for heating box]

[0049] 16. User interface (GUI) 17. C02valve

[0050] 18. Heating pad [for incubator casing]

[0051] 19. Driver board [for pumps]

[0052] 20. Waste container

[0053] 21. Electrical connection [for valve driver board - microcontroller connection]

[0054] 22. Tubing [for solution - pump - solenoid valve connections]

[0055] 23. Tubing [for manifold - flow box connection]

[0056] 24. Cell and analysis solutions

[0057] 25. Electrical connection [for microcontroller - heating box connection]

[0058] 26. Electrical connection [for microcontroller - incubator casing connection]

[0059] 27. SPE (Screen-Printed Electrode)

[0060] 271. Working electrode

[0061] 272. Reference electrode

[0062] 273. Counter electrode

[0063] 28. Incubator casing

[0064] 29. Flow box

[0065] 30. Tubing [for flow box - waste container connection]

[0066] 31. Pump

[0067] 32. Sample preparation apparatus

[0068] 33. Top layer of the flow box

[0069] 331. Inlet [for the flow box]

[0070] 332. Outlet [for the flow box]

[0071] 333. Chamber [for the flow box]

[0072] 34. Bottom layer of the flow box

[0073] 341. Slot [for the flow box]

[0074] 35. Top cover of the flow box

[0075] 351. Hole [for the flow box]

[0076] 352. Central hole [for the flow box]

[0077] 36. Bottom cover of the flow box 361. Slot [for the flow box]

[0078] 37. Electrical connection [for pump driver board - microcontroller connection]

[0079] 38. UVC light source

[0080] 39. USB cable [for potentiostat connection]

[0081] 40. Fan [for heating box]

[0082] 41. Fan [for incubator casing]

[0083] 42. Display

[0084] 43. Electrical Connection [for display microcontroller connection]

[0085] 44. Microcontroller [for display]

[0086] 45. USB cable [for microcontroller]

[0087] 46. Sandwich structure

[0088] 47. Bottom layer of sample preparation apparatus

[0089] 471 Slot [for sample preparation apparatus]

[0090] 48. Top layer of sample preparation apparatus

[0091] 481 Chamber [for sample preparation apparatus]

[0092] 49. Top cover of sample preparation apparatus

[0093] 491 Hole [for sample preparation apparatus]

[0094] 50. Bottom cover of sample preparation apparatus

[0095] 501 Slot [for sample preparation apparatus]

[0096] Detailed Description of the Invention

[0097] The electrochemical analysis platform described in this invention consists of a Microfluidic Module, which includes a flow box (29) and pump (31); an Incubator Module that provides a suitable environment for cell culture; an Electrochemical Testing Module with integrated SPEs (27) and a potentiostat (2); and a User Interface Module (Figure 1).

[0098] The invention is an electrochemical analysis platform integrated into an incubator casing (28) that facilitates cell-based electrochemical analyses using SPE (27) technology.

[0099] The invention provides: • Reduction of the effects of exogenous factors such as pH, temperature, and electrolyte salt concentration on live cells incubated on the electrode surface,

[0100] • Prevention of external influences on the cells located on the electrode surface,

[0101] • High-accuracy cell-based electrochemical measurements by ensuring cell viability and proliferation under incubator conditions.

[0102] The invention is composed of four main modules (Figure 1):

[0103] 1. Microfluidic Module: This module includes a flow box (29) where SPEs (27) interact with cell and analysis solutions (24) and a pump (31) that delivers these solutions (24) to the flow box (29).

[0104] 2. Incubator Module: This module comprises an incubator casing (28) that ensures SPE (27) based measurements are conducted in an environment suitable for cell proliferation.

[0105] 3. Electrochemical Testing Module: This module includes SPEs (27) for cell-based tests and a potentiostat (2) for performing the measurements.

[0106] 4. User Interface Module: This module consists of a user interface or GUI (16) for controlling device connections, cell tests, pump (31) and incubator settings.

[0107] Incubator Integration: The invention integrates an Incubator Module for electrochemical analyses. The incubator casing (28) ensures that cells are analyzed under optimal conditions. Cells are preserved in a stable environment with precise temperature, carbon dioxide levels, and sterilization conditions. This ensures cell viability and proliferation, allowing the collection of highly accurate electrochemical data.

[0108] Microfluidic Pump: The invention facilitates the delivery of fresh culture medium to the cells through a pump (31). This feature preserves the natural behavior of the cells, enabling analysis in a stable environment without exposure to external factors. By nourishing and maintaining the cells under natural conditions, the reliability of electrochemical analyses is enhanced, ensuring the scientific accuracy and validity of the data.

[0109] Single Interface Control and Automation: The invention allows for the control of all components, including the potentiostat (2), the pump (31), and the incubator casing (28), via a single user interface (16) (Figure 3), enabling the automated execution of protocols. This approach minimizes user error, enhances the reliability and reproducibility of electrochemical analyses, and improves result accuracy. The system ensures the efficient and error-free execution of complex analyses and is fully compatible with all commercially available potentiostats (2). Ease of Sample Preparation: The sample preparation apparatus (32) of the platform simplifies the seeding of cell samples onto the SPE (27) surface for subsequent electrochemical analyses. This apparatus ensures robust and consistent cell seeding on the SPE (27) surface within a commercial incubator streamlining and controlling the sample preparation process, thereby accelerating laboratory studies.

[0110] The sample preparation apparatus (32) ensures that the surface of the SPEs (27) used in electrochemical analyses remains in direct contact with the incubator environment while fully covering the electrical regions. This design protects the sensitive areas of the SPEs (27), resulting in more reliable electrochemical analysis results. The surface of the SPE (27) is the only part exposed to the incubator environment, effectively preventing any potential effects of the incubator environment on the electrical regions of the SPE (27).

[0111] The invention operates by integrating four distinct modules to operate simultaneously. All commands for the system's modules are transmitted through the user interface (16). Initially, cells are incubated on the surface of the SPE (27) for a user-defined period in a commercial incubator using the sample preparation apparatus (32), ensuring proper adhesion of the cells on the SPE (27) surface. The sample preparation apparatus (32) ensures that only the SPE (27) surface comes into contact with the incubator environment, while the remaining regions of the SPE (27) are fully shielded. This ensures that only the three-electrode configuration, consisting of the working (271), reference (272), and counter electrodes (273) of the SPE (27), is exposed to the incubator environment, thus preventing any potential effects of the incubator environment on the electrical regions of the SPE (27).

[0112] Before conducting electrochemical tests in the system, the incubator module is activated via the user interface (16), establishing the optimal conditions (37°C, 5% CO2) necessary for cell maintenance. Subsequently, the SPEs (27) are removed from the sample preparation apparatus (32) and placed in the flow box (29). The SPE (27) is then connected to a manifold (9) via tubing (23) integrated within the flow box (29).

[0113] Next, the SPE (27) integrated into the flow box (29) is placed inside the incubator casing (28). The SPE (27) is connected to the potentiostat (2) via an SPE connector (6), with the connection points between the SPE (27) and the SPE connector (6) positioned outside the incubator casing (28) to prevent any influence of the incubator environment on the electrical connections. Cell and analysis solutions (24) are delivered to the SPE (27) surface through tubing (22) connected to the pumps (31) in the Microfluidic Module and tubing (23) linked to the flow box (29). Finally, the operator selects the appropriate measurement technique through the user interface (16) to perform the tests. The acquired data and graphs are stored through the user interface (16). After the measurements are completed, cleaning solutions are delivered to the SPE (27) surface via the pump (31) system, ensuring thorough cleaning of all surfaces that contacted with the cell and analysis solutions (24).

[0114] 1. Microfluidic Module

[0115] The flow box (29) consists of the top (33) and bottom (34) layers made of biocompatible and elastic materials (Figure 2). The design of the flow box (29), specifically tailored for SPEs (27), ensures that the cell medium placed on the SPE (27) uniformly covers its surface. In the top layer (33) of the flow box (29), a circular chamber (333) is aligned with the three-electrode configuration of the working (271), reference (272), and counter electrodes (273). Two channels connect to this chamber (333) via inlet (331) and outlet (332), facilitating the introduction and removal of cell and analysis solutions (24) via tubing (23). The bottom layer (34) of the flow box (29) includes a slot (341) specifically designed to secure the SPE (27).

[0116] The top cover (35) of the flow box (29), as illustrated in Figure 2, includes two holes (351) aligned with the inlet (331)and outlet (332) in the top layer (33). A larger central hole (352) is aligned with the circular chamber (333) in the top layer (33). The purpose of this central hole (352) is to prevent the thinner upper top (33) from bending under pressure applied by the top cover (35). The bottom cover (36) of the flow box (29) includes a slot (361) where the bottom layer (34) of the flow box (29) is located. Aligning components within the covers (35, 36) ensures that the layers (33, 34) are automatically aligned within the flow box (29) without requiring operator intervention. Small screws are positioned at the junctions of the top and bottom covers (35, 36), allowing the flow box (29) to be easily and securely assembled. Once the measurement process is completed, the flow box can be easily disassembled. This mechanism ensures that SPEs (27) are securely placed in the flow box (29), and the layers (33, 34) compressed by the covers (35, 36) create a leak-proof seal over the SPE (27) surface.

[0117] Cell and analysis solutions (24) were delivered to the chamber (333) inside the flow box (29) via a system comprising of n pumps (31), a pump driver board (19), an n-inlet single-outlet manifold (9), n valves (8), n valve driver boards (7), and a microcontroller (1). The pump (31), driver board (19), and microcontroller (1) are interconnected through an electrical connection (37). Communication between the microcontroller (1) and the driver board (19) is facilitated via a data transfer protocol.

[0118] Different voltage values generated by the microcontroller (1) are transmitted to the pumps (31) via the driver board (19), enabling the flow rates of the pumps (31) to be adjusted by varying these voltage values. The driver board (19) independently controls n pumps (31).. By adjusting the flow rates and durations, the required volume for the system is supplied. The valves (8) are controlled by the microcontroller (1) through their dedicated drivers (7). A separate driver board (7) is used for each valve (8) connected to the manifold (9). In total, n driver boards (7) are utilized to enable the opening and closing of the valves (8) with the control of the microcontroller (1).

[0119] The manifold (9) features n inlets and a single outlet, enabling only the desired solution among n different solutions to reach the flow box (29). When the valve (8) for a specific manifold inlet is opened, the pump (31) connected to this particular inlet is activated, ensuring that the solutions are delivered to the flow box (29) only from the open inlet. The valve (8), driver board (7), and the microcontroller (1) are interconnected via an electrical connection (21).

[0120] Cell and analysis solutions (24) are stored in a heating box (5), where a heating pad (15) and a CO2valve (17) maintain the solutions at incubator conditions (e.g., 37°C, 5% CO2). This setup prevents external factors from impacting the temperature and pH of the solutions, thereby eliminating any potential adverse effects on the cells.

[0121] The temperature and CO2levels in the heating box (5) are monitored using a temperature sensor (14) and a CO2sensor (10), both connected to a microcontroller (3). The heating pad (15) and the CO2valve (17) are controlled by the microcontroller (3) through a driver board (12). The heating box (5) is linked to the microcontroller (3) via an electrical connection (25).

[0122] Additionally, this module also includes a waste container (20) designed to collect waste solutions discharged from the flow box (29), which is connected to the flow box (29) through tubing (30).

[0123] 2. Incubator Module:

[0124] The Incubator Module ensures the optimal conditions for cells (e.g., 37°C, 5% CO2). Within the incubator casing (28), continuous readings are obtained via the temperature sensor (11) integrated into the module through the microcontroller (4). The microcontroller (4) incorporates a control algorithm linked to a feedback mechanism, maintaining the stable conditions required for cell proliferation. The incubator casing (28) is connected to the microcontroller (4) through an electrical connection (26).

[0125] In the invention, as the SPEs (27) are continuously immersed in solution, there is no need for humidity control. The required temperature for the cells to remain at the appropriate level (e.g., 37°C) is maintained by the heat pad (18). The temperature sensor (11) is employed to regulate the internal temperature of the incubator casing (28). Positioned near the SPE (27), the temperature sensor (11) ensures continuous monitoring of the temperature in the vicinity of the cells. Connected to the microcontroller (4), the temperature sensor (11) provides real-time readings. Through the microcontroller's (4) control algorithm, changes in temperature are detected from the sensor (11) readings, and adjusts the heat source accordingly by turning it on or off. This mechanism ensures that the temperature remains constant.

[0126] The microcontroller (3) control algorithm for the heating box (5) manages the regulation of both temperature and CO2levels, differing from the control algorithm used for the incubator casing (28).

[0127] By integrating the CO2tank into the heating box (5), a medium with an appropriate pH for the cells is achieved and delivered to the SPE (27) surface from the heating box (5) to the flow box (29) via the appropriate tubing (22, 23). The CO2concentration is monitored using the CO2sensor (10) connected to the microcontroller (3). If the CO2level drops below a specified threshold (e.g., 5%), the microcontroller (3) activates the CO2valve (17) to allow CO2flow from the tank into the heating box (5). Conversely, if the CO2level exceeds the specified threshold, the CO2valve (17) is closed by the microcontroller (3) to stop the CO2flow. To address potential contamination in the incubator environment, sterilization is performed using the Ultraviolet C (UVC) light source (38).

[0128] The heat pads (15, 18) and the CO2valve (17) are controlled via driver boards (12, 13), which supply the necessary voltage and current values for their functioning. The driver boards (12, 13) are connected to the microcontrollers (3, 4), enabling control of the heat pads (15, 18) and the CO2valve (17). To ensure uniform temperature distribution, fans (40, 41) powered by the microcontrollers (3, 4) are installed within the incubator casing (28) and the heating box (5).

[0129] Feedback Mechanism

[0130] The feedback mechanism utilized in the invention is based on a PID (Proportional Integral Derivative) control system, which is widely used in electronic devices, mechanical systems, and industrial control applications (Knospe 2006; Johnson and Moradi 2005). Utilizing feedback, the platform-generated signal is compared to the input signal, and the error difference (i.e., the deviation between the target state and the actual system state is determined.

[0131] As a result, the microcontrollers (3, 4) process signals through the PID system to minimize the error margin and maintain the target temperature (e.g., 37°C) and CO2level (e.g., 5%) at their desired values. The system's temperature and CO2parameters are continuously monitored by sensors (10, 11, 14) and transmitted to the microcontrollers (3, 4). The measured temperature and CO2values from the sensors (10, 11, 14) are compared with the target values to calculate an error difference. The integral and derivative of this error are then computed to determine the appropriate control values.

[0132] These control values are transmitted to the driver boards (12, 13), which operate the heating pads (15, 18) and the CO2valve (17). The heating pads (15, 18) and CO2valve (17) adjust the temperature and CO2levels, which are subsequently remeasured by the temperature sensors (11, 14) and the CO2sensor (10). The updated values are sent back to the microcontrollers (3, 4), forming a continuous feedback loop. This loop persists until the system achieves stabilization at the optimal parameters.

[0133] Additionally, the invention includes a display (42) that independently presents the incubator parameters for the incubator casing (28) and the heating box (5). The display (42) is controlled by a microcontroller (44) connected via an electrical connection (43). System parameters generated by the microcontrollers (3, 4) are transmitted to a computer and subsequently relayed via a USB cable (45) to the microcontroller (44) for visualization on the display (42).

[0134] 3. Electrochemical Testing Module

[0135] The Electrochemical Testing Module consists of the SPE (27) and the potentiostat (2). The SPE (27) is connected to the potentiostat (2) through an SPE connector (6).

[0136] 4. Graphical User Interface (GUI) Module

[0137] The Graphical User Interface [GUI (16)] manages the Microfluidic Module, Incubator Module, and Electrochemical Testing Module. A serial communication algorithm facilitates communication between the GUI (16) and the microcontrollers (1, 3, 4), enabling the GUI (16) to regulate the flow rate and duration of the pumps (31). Similarly, sensor data from the temperature sensors (11, 14) and the CO2sensor (10) are continuously transmitted from the microcontrollers (3, 4) to the GUI (16) in realtime via the serial communication protocol. Live readings from the sensors in the incubator casing (28) and the heating box (5) are displayed on the GUI (16). Additionally, measurement results obtained from the potentiostat (2) in the Electrochemical Testing Module are graphically presented on the GUI (16) using the serial communication protocol.

[0138] Measurement results can be stored in a specified location on a computer through the GUI (16) and can also be accessed and visualized from previously saved data.

[0139] Components of the System GUI

[0140] The system GUI (16) consists of six main components: 1. Connection: The potentiostat (2) connects to the computer via a USB cable (39). The device connection is established by clicking the "Connect" icon. Alternatively, the potentiostat (2) can connect to the computer via Bluetooth, eliminating the need for a USB cable (39). By selecting the "Select" icon, the user can choose from various measurement methods, such as Differential Pulse Voltammetry (DPV) or Cyclic Voltammetry (CV).

[0141] 2. Device Status: The "Device Status" section of the GUI (16) shows real-time measurement values from the potentiostat (2). The "Status" icon provides information on whether the connection between the computer and the device has been successfully established.

[0142] 3. Measurement: In the "Measurement" section, clicking the "Start Measurement" icon begins the measurement process, which is completed within the specified time frame. The "Load" icon enables previously conducted test results to be uploaded and displayed, while the "Save" icon allows the measurement data to be stored in a selected file.

[0143] 4. Graph: The "Graph" section creates graphs using the values obtained during the measurement process. Users can save these graphs to a folder by clicking the "Export" icon. Furthermore, after completing a measurement, users can clear all displayed graphs by clicking the "Clear AH" icon, preparing the interface ready for a new measurement.

[0144] 5. Incubator: In the incubator casing (28), users can set the temperature and CO2concentration of the cell environment. The system begins operation upon pressing the "Start" icon. In emergencies or when the system needs to be stopped, pressing the "Stop" icon ceases all operations. The incubator display section provides real-time updates on the temperature and CO2concentration within both the incubator casing (28) and the heating box (5).

[0145] 6. Pump Settings: In this section, clicking the "Open Settings" icon enables the user to adjust the volume and flow rate of the fluid entering the flow box (29). With the use of n different pumps (31), n different fluids can be introduced to or removed from the cell environment on the SPE (27) surface.

[0146] Sample preparation apparatus (32):

[0147] For accurate electrochemical measurements, cells must adhere to the SPE (27) surface in a healthy and stable manner. If cells detach from the surface during the test, the reliability of the data produced by the platform diminishes. Therefore, before each test, cells are incubated on the SPE (27) surface to ensure firm adhesion. After seeding the cells, the SPEs (27) are placed in a commercial incubator. However, the electrical regions of the SPE (27), such as the area where it connects to the SPE connector (6), may be negatively affected by the incubator environment. To address this, the invention incorporates a sample preparation apparatus (32). This apparatus (32) includes an opening that exposes only the SPE surface where the cells will be incubated, ensuring the remaining regions of the SPE (27) stay covered. Through this opening, the operator can pipette and seed the cells.

[0148] The apparatus consists of two elastic layers (47, 48). The bottom layer (47) includes a slot (471) where the SPE (27) is positioned, while the top layer (48) features a chamber (481) aligned with the electrode area of the SPE (27) to facilitate the incubating cells. These layers (47, 48) are stacked to form a sandwich structure (46). Cells are seeded onto the electrode surface through the chamber (481) by pipetting. The elastic layers (47, 48) are compressed with two covers (49, 50) to ensure the SPE (27) surface remains sealed. The top cover (49) has a hole (491) aligned with the chamber (481) in the top layer (48), while the bottom cover (50) includes a slot (501) for positioning the bottom layer (47).

[0149] As illustrated in Figure 3, the sample preparation apparatus (32) ensures that only the electrode regions of the SPE (27) are exposed. Cells seeded on the electrode surface are incubated in the commercial incubator with the sample preparation apparatus (32). Before testing, the SPEs (27) are removed from the sample preparation apparatus (32) and integrated into the Microfluidic Module.

Claims

CLAIMS1. An electrochemical analysis platform designed to observe the cellular behaviors of live cells on a screen-printed electrode (SPE) (27) using electrochemical methods, while maintaining in-vivo conditions within an incubator environment with controlled carbon dioxide (CO2) and temperature parameters. The invention ensures healthy cell proliferation without exposure to external factors during the application of these methods, comprising• An incubator casing (28) designed to maintain optimal conditions for cell proliferation during measurements conducted on the screen-printed electrode (SPE) (27);• A flow box (29) to facilitate interaction between SPEs (27) and cell and analysis solutions (24);• A sample preparation apparatus (32) that ensures the electrode surface of the SPE (27) remains exposed to the incubator environment, while fully covering its other regions for protection;• A pump (31) to regularly deliver fresh cell and analysis solutions (24) to the cells through the flow box (29);• A single user interface (16) to control all components, including cell tests, pump (31) settings, and incubator settings;• Microcontrollers (3, 4) equipped with temperature sensors (11, 14) and a CO2sensor (10) for continuous monitoring;• A heating pad (18) to maintain the incubator environment at the appropriate temperature;• A temperature sensor (14) to ensure the solutions are maintained at suitable temperatures;• A CO2valve (17) connected to any CO2source to provide the required CO2for maintaining the incubator environment at the appropriate CO2concentration;• A CO2sensor (10) to maintain the culture medium at an appropriate pH value;• A UVC light source (38) for sterilization to prevent contamination in the incubator environment.

2. An electrochemical analysis platform according to claim 1, characterized by including tubings (22, 23) in the microfluidic pump (31) to deliver fresh culture medium to the cells on the electrode surface.

3. An electrochemical analysis platform according to claim 1, characterized by a flow box (29) consisting of biocompatible and elastic materials forming a top layer (33) and a bottom layer (34).

4. An electrochemical analysis platform according to claim 1, characterized by a circular chamber (333) in the top layer (33) of the flow box (29) aligned with the electrodes on the SPE (27), and channels with inlet and outlet (331, 332) for delivering and removing cell and analysis solutions (24) via tubing (23).

5. An electrochemical analysis platform according to claim 1, characterized by including a slot (341) in the bottom layer (34) of the flow box (29) for accommodating the SPE (27).

6. An electrochemical analysis platform according to claim 1, characterized by including inlet and outlet (331, 332) in the top layer (33) of the flow box (29).

7. A flow box (29) according to claim 1, characterized by having a top cover (35) with a central hole (352) aligned with the chamber (333) in the top layer (33) and two holes (351) aligned with the inlet (331) and outlet (332) in the top layer (33).

8. An electrochemical analysis platform according to claim 1, characterized by including a slot (361) in the bottom cover (36) of the flow box (29) for accommodating the bottom layer (34) of the flow box (29).

9. An electrochemical analysis platform according to claim 1, characterized by including n pumps (31) and pump driver boards (19), an n-inlet and single-outlet manifold (9), n valves (8) with n driver boards (7), and a microcontroller (1).

10. An electrochemical analysis method for observing the cellular behaviors of live cells on a SPE (27) using electrochemical methods while maintaining in-vivo conditions in an incubator environment with CO2and temperature parameters, ensuring healthy proliferation of cells without exposure to external factors, and compatible with any potentiostat (2) for conducting cell-based tests, comprising the steps of:• Incubating cells on the electrode surface using a sample preparation apparatus (32) within a commercial incubator for a user-defined duration to ensure cell adhesion on the electrode surface;• Exposing only the electrode surface to the incubator environment while preventing any potential effects of the incubator environment on the electrical regions of the SPE (27);• Activating the incubator module through the user interface (16) before electrochemical tests and achieving the optimum conditions required for cells;• Placing SPEs (27) removed from the sample preparation apparatus (32) into the flow box (29);• Connecting the SPE (27) in the flow box (29) to a manifold (9) via tubing (23);• Integrating the SPE (27) placed in the flow box (29) into the incubator casing (28);• Connecting the SPE (27) to the potentiostat (2) via an SPE connector (6);• Delivering cell and analysis solutions (24) from a heating box (5) to the surface of the SPE (27) through tubing (22) connected to the Microfluidic Module pumps (31) and tubing (23) connected to the flow box (29);• Selecting the appropriate measurement technique through the user interface (16) and conducting the tests;• Collecting waste solutions in a waste container (20).

11. An electrochemical analysis method according to claim 10, characterized by maintaining optimum conditions for cells, such as 37°C and 5% CO2.

12. An electrochemical analysis method according to claim 10, characterized by recording the obtained data and graphs through the user interface (16).

13. An electrochemical analysis method according to claim 10, characterized by delivering cleaning solutions to the electrode surface of the SPE (27) using the pump (31) system after measurements are completed and cleaning all surfaces in contact with the solutions.

14. An electrochemical analysis platform according to claim 1, characterized by including a display (42) that shows the incubator parameters separately for the incubator casing (28) and the heating box (5).

15. An electrochemical analysis platform according to claim 1, characterized by the presence of fans (40, 41) powered by microcontrollers (3, 4) within the incubator casing (28) and the heating box (5) to ensure homogeneous temperature distribution.

16. An electrochemical analysis platform according to claim 1, characterized by the sample preparation apparatus (32) having an opening that exposes only the SPE (27) surface where cells are incubated.

17. An electrochemical analysis platform according to claim 16, characterized by including a bottom layer (47) and a top layer (48) inside the sample preparation apparatus (32), which are compressed by a top cover (49) and a bottom cover (50) to provide a seal to the SPE (27) surface.

18. An electrochemical analysis platform according to claim 17, characterized by the bottom layer (47) having a slot (471) where the SPE (27) is positioned.

19. An electrochemical analysis platform according to claim 17, characterized by the top layer (48) having a chamber (481) where cells is incubated and which aligns with the electrode region of the SPE (27).

20. An electrochemical analysis platform according to claim 17, characterized by including a sandwich structure (46) formed by stacking the bottom layer (47) and the top layer (48).

21. An electrochemical analysis platform according to claim 17, characterized by the top cover (49) having a hole (491) that aligns with the chamber (481) in the top layer (48).

22. An electrochemical analysis platform according to claim 17, characterized by the bottom cover (50) having a slot (501) where the bottom layer (47) is positioned.

23. An electrochemical analysis platform according to claim 1, characterized in that it includes a waste container (20) where the solutions coming out of the flow box (29) and becoming waste are collected.

24. An electrochemical analysis platform according to claim 23, characterized in that the waste container (20) is connected to the flow box (29) by a tubing (30).

Images